CN111087463B - Recombinant human III-type collagen and prokaryotic expression method thereof - Google Patents
Recombinant human III-type collagen and prokaryotic expression method thereof Download PDFInfo
- Publication number
- CN111087463B CN111087463B CN201911384510.2A CN201911384510A CN111087463B CN 111087463 B CN111087463 B CN 111087463B CN 201911384510 A CN201911384510 A CN 201911384510A CN 111087463 B CN111087463 B CN 111087463B
- Authority
- CN
- China
- Prior art keywords
- gene
- recombinant
- type iii
- gly
- iii collagen
- 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
- 238000000034 method Methods 0.000 title claims abstract description 13
- 230000009465 prokaryotic expression Effects 0.000 title claims abstract description 9
- 102000008186 Collagen Human genes 0.000 title description 26
- 108010035532 Collagen Proteins 0.000 title description 26
- 229920001436 collagen Polymers 0.000 title description 26
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 46
- 102000001187 Collagen Type III Human genes 0.000 claims abstract description 28
- 108010069502 Collagen Type III Proteins 0.000 claims abstract description 28
- 239000013604 expression vector Substances 0.000 claims abstract description 20
- 238000003259 recombinant expression Methods 0.000 claims abstract description 18
- 230000014509 gene expression Effects 0.000 claims abstract description 13
- 230000006698 induction Effects 0.000 claims abstract description 9
- 108020004705 Codon Proteins 0.000 claims abstract description 7
- 238000013461 design Methods 0.000 claims abstract description 6
- 102000004190 Enzymes Human genes 0.000 claims abstract description 5
- 108090000790 Enzymes Proteins 0.000 claims abstract description 5
- 238000005520 cutting process Methods 0.000 claims abstract description 5
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 238000005457 optimization Methods 0.000 claims abstract description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 20
- 241000894006 Bacteria Species 0.000 claims description 16
- 230000001939 inductive effect Effects 0.000 claims description 11
- 239000013612 plasmid Substances 0.000 claims description 11
- 230000015572 biosynthetic process Effects 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 7
- 238000003786 synthesis reaction Methods 0.000 claims description 7
- 238000010276 construction Methods 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 230000035939 shock Effects 0.000 claims description 6
- 241001052560 Thallis Species 0.000 claims description 4
- 238000003776 cleavage reaction Methods 0.000 claims description 4
- 239000013613 expression plasmid Substances 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 4
- 230000007017 scission Effects 0.000 claims description 4
- 230000001131 transforming effect Effects 0.000 claims description 4
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 claims 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 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 241000588724 Escherichia coli Species 0.000 abstract description 5
- 238000010367 cloning Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- 239000012528 membrane Substances 0.000 description 14
- 108010014614 prolyl-glycyl-proline Proteins 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 11
- 239000002033 PVDF binder Substances 0.000 description 9
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 9
- 239000000872 buffer Substances 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 8
- 239000012154 double-distilled water Substances 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 238000012546 transfer Methods 0.000 description 8
- 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 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- BRPMXFSTKXXNHF-IUCAKERBSA-N (2s)-1-[2-[[(2s)-pyrrolidine-2-carbonyl]amino]acetyl]pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)CNC(=O)[C@H]1NCCC1 BRPMXFSTKXXNHF-IUCAKERBSA-N 0.000 description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 6
- 150000001413 amino acids Chemical group 0.000 description 6
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 6
- 210000003491 skin Anatomy 0.000 description 6
- OOCFXNOVSLSHAB-IUCAKERBSA-N Gly-Pro-Pro Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 OOCFXNOVSLSHAB-IUCAKERBSA-N 0.000 description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 5
- 108010077515 glycylproline Proteins 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 108010079364 N-glycylalanine Proteins 0.000 description 4
- 239000001110 calcium chloride Substances 0.000 description 4
- 229910001628 calcium chloride Inorganic materials 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- JYPCXBJRLBHWME-UHFFFAOYSA-N glycyl-L-prolyl-L-arginine Natural products NCC(=O)N1CCCC1C(=O)NC(CCCN=C(N)N)C(O)=O JYPCXBJRLBHWME-UHFFFAOYSA-N 0.000 description 4
- 238000010369 molecular cloning Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 108010029020 prolylglycine Proteins 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 239000012224 working solution Substances 0.000 description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 102000012422 Collagen Type I Human genes 0.000 description 3
- 108010022452 Collagen Type I Proteins 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- KKBWDNZXYLGJEY-UHFFFAOYSA-N Gly-Arg-Pro Natural products NCC(=O)NC(CCNC(=N)N)C(=O)N1CCCC1C(=O)O KKBWDNZXYLGJEY-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 3
- UGTHTQWIQKEDEH-BQBZGAKWSA-N L-alanyl-L-prolylglycine zwitterion Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UGTHTQWIQKEDEH-BQBZGAKWSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 3
- 108010047495 alanylglycine Proteins 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 108010087846 prolyl-prolyl-glycine Proteins 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 108010026333 seryl-proline Proteins 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- SCAKQYSGEIHPLV-IUCAKERBSA-N (4S)-4-[(2-aminoacetyl)amino]-5-[(2S)-2-(carboxymethylcarbamoyl)pyrrolidin-1-yl]-5-oxopentanoic acid Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O SCAKQYSGEIHPLV-IUCAKERBSA-N 0.000 description 2
- NBTGEURICRTMGL-WHFBIAKZSA-N Ala-Gly-Ser Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O NBTGEURICRTMGL-WHFBIAKZSA-N 0.000 description 2
- CKRUHITYRFNUKW-WDSKDSINSA-N Glu-Asn-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O CKRUHITYRFNUKW-WDSKDSINSA-N 0.000 description 2
- PYTZFYUXZZHOAD-WHFBIAKZSA-N Gly-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)CN PYTZFYUXZZHOAD-WHFBIAKZSA-N 0.000 description 2
- PUUYVMYCMIWHFE-BQBZGAKWSA-N Gly-Ala-Arg Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PUUYVMYCMIWHFE-BQBZGAKWSA-N 0.000 description 2
- VXKCPBPQEKKERH-IUCAKERBSA-N Gly-Arg-Pro Chemical compound NC(N)=NCCC[C@H](NC(=O)CN)C(=O)N1CCC[C@H]1C(O)=O VXKCPBPQEKKERH-IUCAKERBSA-N 0.000 description 2
- CAVKXZMMDNOZJU-UHFFFAOYSA-N Gly-Pro-Ala-Gly-Pro Natural products C1CCC(C(O)=O)N1C(=O)CNC(=O)C(C)NC(=O)C1CCCN1C(=O)CN CAVKXZMMDNOZJU-UHFFFAOYSA-N 0.000 description 2
- JYPCXBJRLBHWME-IUCAKERBSA-N Gly-Pro-Arg Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O JYPCXBJRLBHWME-IUCAKERBSA-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
- UCBPDSYUVAAHCD-UWVGGRQHSA-N Leu-Pro-Gly Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UCBPDSYUVAAHCD-UWVGGRQHSA-N 0.000 description 2
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 2
- MMJJFXWMCMJMQA-STQMWFEESA-N Phe-Pro-Gly Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)C1=CC=CC=C1 MMJJFXWMCMJMQA-STQMWFEESA-N 0.000 description 2
- KIZQGKLMXKGDIV-BQBZGAKWSA-N Pro-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 KIZQGKLMXKGDIV-BQBZGAKWSA-N 0.000 description 2
- BNBBNGZZKQUWCD-IUCAKERBSA-N Pro-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H]1CCCN1 BNBBNGZZKQUWCD-IUCAKERBSA-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
- DXTOOBDIIAJZBJ-BQBZGAKWSA-N Pro-Gly-Ser Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CO)C(O)=O DXTOOBDIIAJZBJ-BQBZGAKWSA-N 0.000 description 2
- BGWKULMLUIUPKY-BQBZGAKWSA-N Pro-Ser-Gly Chemical compound OC(=O)CNC(=O)[C@H](CO)NC(=O)[C@@H]1CCCN1 BGWKULMLUIUPKY-BQBZGAKWSA-N 0.000 description 2
- RHAPJNVNWDBFQI-BQBZGAKWSA-N Ser-Pro-Gly Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O RHAPJNVNWDBFQI-BQBZGAKWSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 2
- 108010069926 arginyl-glycyl-serine Proteins 0.000 description 2
- 108010047857 aspartylglycine Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 108010025801 glycyl-prolyl-arginine Proteins 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- CUVSTAMIHSSVKL-UWVGGRQHSA-N (4s)-4-[(2-aminoacetyl)amino]-5-[[(2s)-6-amino-1-(carboxymethylamino)-1-oxohexan-2-yl]amino]-5-oxopentanoic acid Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CN CUVSTAMIHSSVKL-UWVGGRQHSA-N 0.000 description 1
- 101150084750 1 gene Proteins 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- ZVFVBBGVOILKPO-WHFBIAKZSA-N Ala-Gly-Ala Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O ZVFVBBGVOILKPO-WHFBIAKZSA-N 0.000 description 1
- WMYJZJRILUVVRG-WDSKDSINSA-N Ala-Gly-Gln Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O WMYJZJRILUVVRG-WDSKDSINSA-N 0.000 description 1
- VGPWRRFOPXVGOH-BYPYZUCNSA-N Ala-Gly-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)NCC(O)=O VGPWRRFOPXVGOH-BYPYZUCNSA-N 0.000 description 1
- BLIMFWGRQKRCGT-YUMQZZPRSA-N Ala-Gly-Lys Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN BLIMFWGRQKRCGT-YUMQZZPRSA-N 0.000 description 1
- NIZKGBJVCMRDKO-KWQFWETISA-N Ala-Gly-Tyr Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NIZKGBJVCMRDKO-KWQFWETISA-N 0.000 description 1
- PNALXAODQKTNLV-JBDRJPRFSA-N Ala-Ile-Ala Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O PNALXAODQKTNLV-JBDRJPRFSA-N 0.000 description 1
- DVJSJDDYCYSMFR-ZKWXMUAHSA-N Ala-Ile-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O DVJSJDDYCYSMFR-ZKWXMUAHSA-N 0.000 description 1
- 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 1
- CQJHFKKGZXKZBC-BPNCWPANSA-N Ala-Pro-Tyr Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 CQJHFKKGZXKZBC-BPNCWPANSA-N 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- AQPVUEJJARLJHB-BQBZGAKWSA-N Arg-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CCCN=C(N)N AQPVUEJJARLJHB-BQBZGAKWSA-N 0.000 description 1
- HQIZDMIGUJOSNI-IUCAKERBSA-N Arg-Gly-Arg Chemical compound N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O HQIZDMIGUJOSNI-IUCAKERBSA-N 0.000 description 1
- AUFHLLPVPSMEOG-YUMQZZPRSA-N Arg-Gly-Glu Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O AUFHLLPVPSMEOG-YUMQZZPRSA-N 0.000 description 1
- HAVKMRGWNXMCDR-STQMWFEESA-N Arg-Gly-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O HAVKMRGWNXMCDR-STQMWFEESA-N 0.000 description 1
- WVNFNPGXYADPPO-BQBZGAKWSA-N Arg-Gly-Ser Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O WVNFNPGXYADPPO-BQBZGAKWSA-N 0.000 description 1
- CTQIOCMSIJATNX-WHFBIAKZSA-N Asn-Gly-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C)C(O)=O CTQIOCMSIJATNX-WHFBIAKZSA-N 0.000 description 1
- GKKUBLFXKRDMFC-BQBZGAKWSA-N Asn-Pro-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O GKKUBLFXKRDMFC-BQBZGAKWSA-N 0.000 description 1
- DBLPNHGKMDHWNZ-UHFFFAOYSA-N Asp Gly Arg Asn Chemical compound OC(=O)CC(N)C(=O)NCC(=O)NC(CCCN=C(N)N)C(=O)NC(CC(N)=O)C(O)=O DBLPNHGKMDHWNZ-UHFFFAOYSA-N 0.000 description 1
- AXXCUABIFZPKPM-BQBZGAKWSA-N Asp-Arg-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O AXXCUABIFZPKPM-BQBZGAKWSA-N 0.000 description 1
- VIRHEUMYXXLCBF-WDSKDSINSA-N Asp-Gly-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O VIRHEUMYXXLCBF-WDSKDSINSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- DZLQXIFVQFTFJY-BYPYZUCNSA-N Cys-Gly-Gly Chemical compound SC[C@H](N)C(=O)NCC(=O)NCC(O)=O DZLQXIFVQFTFJY-BYPYZUCNSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 238000012270 DNA recombination Methods 0.000 description 1
- 108010072062 GEKG peptide Proteins 0.000 description 1
- HHWQMFIGMMOVFK-WDSKDSINSA-N Gln-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(N)=O HHWQMFIGMMOVFK-WDSKDSINSA-N 0.000 description 1
- VSXBYIJUAXPAAL-WDSKDSINSA-N Gln-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CCC(N)=O VSXBYIJUAXPAAL-WDSKDSINSA-N 0.000 description 1
- NLKVNZUFDPWPNL-YUMQZZPRSA-N Glu-Arg-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O NLKVNZUFDPWPNL-YUMQZZPRSA-N 0.000 description 1
- RAUDKMVXNOWDLS-WDSKDSINSA-N Glu-Gly-Ser Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O RAUDKMVXNOWDLS-WDSKDSINSA-N 0.000 description 1
- RFTVTKBHDXCEEX-WDSKDSINSA-N Glu-Ser-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RFTVTKBHDXCEEX-WDSKDSINSA-N 0.000 description 1
- GPSHCSTUYOQPAI-JHEQGTHGSA-N Glu-Thr-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O GPSHCSTUYOQPAI-JHEQGTHGSA-N 0.000 description 1
- QXPRJQPCFXMCIY-NKWVEPMBSA-N Gly-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN QXPRJQPCFXMCIY-NKWVEPMBSA-N 0.000 description 1
- AIJAPFVDBFYNKN-WHFBIAKZSA-N Gly-Asn-Asp Chemical compound C([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)CN)C(=O)N AIJAPFVDBFYNKN-WHFBIAKZSA-N 0.000 description 1
- MHHUEAIBJZWDBH-YUMQZZPRSA-N Gly-Asp-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)CN MHHUEAIBJZWDBH-YUMQZZPRSA-N 0.000 description 1
- PABFFPWEJMEVEC-JGVFFNPUSA-N Gly-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)CN)C(=O)O PABFFPWEJMEVEC-JGVFFNPUSA-N 0.000 description 1
- DHDOADIPGZTAHT-YUMQZZPRSA-N Gly-Glu-Arg Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DHDOADIPGZTAHT-YUMQZZPRSA-N 0.000 description 1
- ZQIMMEYPEXIYBB-IUCAKERBSA-N Gly-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CN ZQIMMEYPEXIYBB-IUCAKERBSA-N 0.000 description 1
- MBOAPAXLTUSMQI-JHEQGTHGSA-N Gly-Glu-Thr Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MBOAPAXLTUSMQI-JHEQGTHGSA-N 0.000 description 1
- HMHRTKOWRUPPNU-RCOVLWMOSA-N Gly-Ile-Gly Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O HMHRTKOWRUPPNU-RCOVLWMOSA-N 0.000 description 1
- LOEANKRDMMVOGZ-YUMQZZPRSA-N Gly-Lys-Asp Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CC(O)=O)C(O)=O LOEANKRDMMVOGZ-YUMQZZPRSA-N 0.000 description 1
- QSQXZZCGPXQBPP-BQBZGAKWSA-N Gly-Pro-Cys Chemical compound C1C[C@H](N(C1)C(=O)CN)C(=O)N[C@@H](CS)C(=O)O QSQXZZCGPXQBPP-BQBZGAKWSA-N 0.000 description 1
- HAOUOFNNJJLVNS-BQBZGAKWSA-N Gly-Pro-Ser Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O HAOUOFNNJJLVNS-BQBZGAKWSA-N 0.000 description 1
- BMWFDYIYBAFROD-WPRPVWTQSA-N Gly-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN BMWFDYIYBAFROD-WPRPVWTQSA-N 0.000 description 1
- OHUKZZYSJBKFRR-WHFBIAKZSA-N Gly-Ser-Asp Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O OHUKZZYSJBKFRR-WHFBIAKZSA-N 0.000 description 1
- ABPRMMYHROQBLY-NKWVEPMBSA-N Gly-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)CN)C(=O)O ABPRMMYHROQBLY-NKWVEPMBSA-N 0.000 description 1
- NWOSHVVPKDQKKT-RYUDHWBXSA-N Gly-Tyr-Gln Chemical compound [H]NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O NWOSHVVPKDQKKT-RYUDHWBXSA-N 0.000 description 1
- GWCJMBNBFYBQCV-XPUUQOCRSA-N Gly-Val-Ala Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O GWCJMBNBFYBQCV-XPUUQOCRSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- DCRODRAURLJOFY-XPUUQOCRSA-N His-Ala-Gly Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(=O)NCC(O)=O DCRODRAURLJOFY-XPUUQOCRSA-N 0.000 description 1
- ZIMTWPHIKZEHSE-UWVGGRQHSA-N His-Arg-Gly Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O ZIMTWPHIKZEHSE-UWVGGRQHSA-N 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- QCBYAHHNOHBXIH-UWVGGRQHSA-N His-Pro-Gly Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)C1=CN=CN1 QCBYAHHNOHBXIH-UWVGGRQHSA-N 0.000 description 1
- LBRCLQMZAHRTLV-ZKWXMUAHSA-N Ile-Gly-Ser Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O LBRCLQMZAHRTLV-ZKWXMUAHSA-N 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- LVTJJOJKDCVZGP-QWRGUYRKSA-N Leu-Lys-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O LVTJJOJKDCVZGP-QWRGUYRKSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- UETQMSASAVBGJY-QWRGUYRKSA-N Lys-Gly-His Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CNC=N1 UETQMSASAVBGJY-QWRGUYRKSA-N 0.000 description 1
- SBQDRNOLGSYHQA-YUMQZZPRSA-N Lys-Ser-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SBQDRNOLGSYHQA-YUMQZZPRSA-N 0.000 description 1
- OOLVTRHJJBCJKB-IHRRRGAJSA-N Met-Tyr-Asp Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N OOLVTRHJJBCJKB-IHRRRGAJSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- ZPPVJIJMIKTERM-YUMQZZPRSA-N Pro-Gln-Gly Chemical compound OC(=O)CNC(=O)[C@H](CCC(=O)N)NC(=O)[C@@H]1CCCN1 ZPPVJIJMIKTERM-YUMQZZPRSA-N 0.000 description 1
- CLNJSLSHKJECME-BQBZGAKWSA-N Pro-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H]1CCCN1 CLNJSLSHKJECME-BQBZGAKWSA-N 0.000 description 1
- VYWNORHENYEQDW-YUMQZZPRSA-N Pro-Gly-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 VYWNORHENYEQDW-YUMQZZPRSA-N 0.000 description 1
- WSRWHZRUOCACLJ-UWVGGRQHSA-N Pro-Gly-His Chemical compound C([C@@H](C(=O)O)NC(=O)CNC(=O)[C@H]1NCCC1)C1=CN=CN1 WSRWHZRUOCACLJ-UWVGGRQHSA-N 0.000 description 1
- FKLSMYYLJHYPHH-UWVGGRQHSA-N Pro-Gly-Leu Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O FKLSMYYLJHYPHH-UWVGGRQHSA-N 0.000 description 1
- FEVDNIBDCRKMER-IUCAKERBSA-N Pro-Gly-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)CNC(=O)[C@@H]1CCCN1 FEVDNIBDCRKMER-IUCAKERBSA-N 0.000 description 1
- XQSREVQDGCPFRJ-STQMWFEESA-N Pro-Gly-Phe Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O XQSREVQDGCPFRJ-STQMWFEESA-N 0.000 description 1
- AFXCXDQNRXTSBD-FJXKBIBVSA-N Pro-Gly-Thr Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O AFXCXDQNRXTSBD-FJXKBIBVSA-N 0.000 description 1
- XYHMFGGWNOFUOU-QXEWZRGKSA-N Pro-Ile-Gly Chemical compound OC(=O)CNC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]1CCCN1 XYHMFGGWNOFUOU-QXEWZRGKSA-N 0.000 description 1
- WIPAMEKBSHNFQE-IUCAKERBSA-N Pro-Met-Gly Chemical compound CSCC[C@@H](C(=O)NCC(=O)O)NC(=O)[C@@H]1CCCN1 WIPAMEKBSHNFQE-IUCAKERBSA-N 0.000 description 1
- FUOGXAQMNJMBFG-WPRPVWTQSA-N Pro-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 FUOGXAQMNJMBFG-WPRPVWTQSA-N 0.000 description 1
- 108010052388 RGES peptide Proteins 0.000 description 1
- IXCHOHLPHNGFTJ-YUMQZZPRSA-N Ser-Gly-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CO)N IXCHOHLPHNGFTJ-YUMQZZPRSA-N 0.000 description 1
- GSCVDSBEYVGMJQ-SRVKXCTJSA-N Ser-Tyr-Asp Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CO)N)O GSCVDSBEYVGMJQ-SRVKXCTJSA-N 0.000 description 1
- 208000028990 Skin injury Diseases 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- TYVAWPFQYFPSBR-BFHQHQDPSA-N Thr-Ala-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)NCC(O)=O TYVAWPFQYFPSBR-BFHQHQDPSA-N 0.000 description 1
- SGAOHNPSEPVAFP-ZDLURKLDSA-N Thr-Ser-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SGAOHNPSEPVAFP-ZDLURKLDSA-N 0.000 description 1
- LFCQXIXJQXWZJI-BZSNNMDCSA-N Tyr-His-His Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)N[C@@H](CC3=CN=CN3)C(=O)O)N)O LFCQXIXJQXWZJI-BZSNNMDCSA-N 0.000 description 1
- PIFJAFRUVWZRKR-QMMMGPOBSA-N Val-Gly-Gly Chemical compound CC(C)[C@H]([NH3+])C(=O)NCC(=O)NCC([O-])=O PIFJAFRUVWZRKR-QMMMGPOBSA-N 0.000 description 1
- YTPLVNUZZOBFFC-SCZZXKLOSA-N Val-Gly-Pro Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N1CCC[C@@H]1C(O)=O YTPLVNUZZOBFFC-SCZZXKLOSA-N 0.000 description 1
- JAKHAONCJJZVHT-DCAQKATOSA-N Val-Lys-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)O)N JAKHAONCJJZVHT-DCAQKATOSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 1
- 108010045023 alanyl-prolyl-tyrosine Proteins 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 108010009111 arginyl-glycyl-glutamic acid Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 108010004073 cysteinylcysteine Proteins 0.000 description 1
- 210000004207 dermis Anatomy 0.000 description 1
- 238000012938 design process Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 108010062266 glycyl-glycyl-argininal Proteins 0.000 description 1
- 108010026364 glycyl-glycyl-leucine Proteins 0.000 description 1
- 108010078326 glycyl-glycyl-valine Proteins 0.000 description 1
- 108010020688 glycylhistidine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 108010081551 glycylphenylalanine Proteins 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 108010003700 lysyl aspartic acid Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- -1 polypropylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000031877 prophase Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 108010020532 tyrosyl-proline Proteins 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 108010000998 wheylin-2 peptide 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
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- 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
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a recombinant human type III collagen and a prokaryotic expression method thereof, wherein the amino acid sequence of the human type III collagen is shown as SEQ ID NO.1, the nucleotide sequence of a coding gene is shown as SEQ ID NO.2, a gene sequence is obtained by codon optimization and design, the gene sequence is inserted between enzyme cutting sites NdeI and XhoI of an expression vector PET30a (+) to construct a recombinant expression vector pET30-1230, competent cells of escherichia coli BL211(DE3) are transformed, positive cloning is selected, and efficient expression is carried out by culture induction.
Description
Technical Field
The invention belongs to the technical field of optimized coding genes, and particularly relates to a recombinant human type III collagen and a prokaryotic expression method thereof.
Background
Collagen is a glycoprotein, which is classified into more than 28 types of collagen according to differences in tissue sites, physiological functions and molecular structures, and the types of collagen, I, II and III, which are the most thoroughly studied, account for the most. Collagen in human skin is type I collagen and type III collagen, wherein the type I collagen is mainly present in adult skin, tendon and bone tissues, the type III collagen is mainly present in infant skin or blood vessel intima and intestinal tract, and the type I collagen and the type III collagen are closely related to the skin injury repair process and repair quality. The III type collagen is a three-strand spiral shape formed by twisting three peptide chains rightward, has excellent biological activity and histocompatibility, can promote fibroblast proliferation of a dermis layer, improves cell activity, is absorbed by fibroblast serving as a raw material for synthesizing the collagen, stimulates cells to synthesize more collagen, fills and repairs damaged and aged skin, reconstructs a reticular structure, enhances the expansion force of the damaged skin, and recovers the elasticity of the skin.
The complex structure of native collagen and the insolubility in water limit the development, utilization and production of native collagen. Collagen products sold in the market at present are all obtained from animal tissues such as pigs, cows, fish and the like, and are further processed by acid, alkali and enzymolysis methods, the biological activity of the collagen products is far lower than that of natural collagen of human bodies, and the collagen products cannot meet the industrial requirements of medicines, foods, cosmetics and the like. With the rapid development of DNA recombination technology, the recombination technology for type III human collagen in the prior art is reported as follows: CN110194795A discloses a recombinant human collagen and application thereof, wherein a section of sequence of a human three-type protein conserved region is selected and repeated for 8 times, the designed sequence is recombined to construct a prokaryotic expression vector pET22b, and the recombinant human collagen which can be efficiently expressed in escherichia coli and is soluble in water is finally obtained through induction expression. CN103122027A discloses a recombinant human collagen and a production method thereof, the structure is a single chain structure, the basic repeating unit is gergapgfrgpaggpngipgekgpagegap which is a human collagen III type peptide segment, the terminal sequence is GPPGPCCGGG which is a human collagen II type peptide segment. The above has the following disadvantages: the patent selects a short segment as a repeating unit, and the short segment is spliced together to express the recombinant human collagen with a long segment, so that the sequence of the human collagen cannot be well copied.
Disclosure of Invention
The invention aims to provide a recombinant human type III collagen with high biological activity and good water solubility, and simultaneously improve the prokaryotic expression efficiency.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
on the one hand, the invention provides a recombinant humanized III type collagen, the amino acid sequence of which is shown in SEQ ID NO. 1.
On the other hand, the invention provides that the nucleotide sequence of the encoding gene of the human type III collagen is shown as SEQ ID NO. 2.
In still another aspect, the invention provides a recombinant expression plasmid pET30-1230 containing the encoding gene of the human type III collagen.
In some embodiments of the invention, the gene encoding human type III collagen is inserted between the cleavage sites NdeI and XhoI of the expression vector PET30a (+).
The invention also provides a recombinant genetic engineering bacterium containing the recombinant expression plasmid pET30-1230, and the host cell of the recombinant genetic engineering bacterium is Escherichia coli BL21(DE 3).
The last aspect of the invention provides a prokaryotic expression method of the human type III collagen, which specifically comprises the following steps:
(1) gene design and synthesis: reversely designing a gene sequence according to the amino acid sequence SEQ ID NO:1 of the human type III collagen, carrying out codon optimization to obtain a gene sequence SEQ ID NO:2, and carrying out whole-gene synthesis according to the gene sequence shown in SEQ ID NO: 2;
(2) construction of recombinant expression vector pET 30-1230: 2, the coding gene of the human III type collagen is inserted between enzyme cutting sites NdeI and XhoI of an expression vector PET30a (+), and is transformed into host bacteria by a heat shock methodE.coliSelecting positive clones from DH5 alpha, and extracting plasmids by adopting a plasmid rapid extraction kit to obtain a recombinant expression vector pET 30-1230;
(3) construction of transformant BL21(DE3)/PET 30-1230: transforming the recombinant expression vector pET30-1230 into competent cells BL21(DE3) through heat shock, and screening to obtain recombinant gene engineering bacteria;
(4) inducing expression: inoculating the obtained recombinant gene engineering bacteria into an M9 culture medium, culturing overnight at 37 ℃, inoculating the obtained recombinant gene engineering bacteria into an M9 culture medium with the inoculation amount of 2%, culturing for 14h at 23 ℃, adding IPTG (0.05 mM) for induction expression, continuously culturing for 12h, and centrifugally collecting thalli.
Adopt the produced beneficial effect of above-mentioned technical scheme to lie in:
the invention
(1) The type III collagen amino acid sequence selected by the invention is longer than other reports, but not repeated expression of specific short human type III collagen amino acid sequence.
(2) The amino acid sequence and the protein space structure of the recombinant human type III collagen obtained by the invention are closer to those of human type III collagen, and the recombinant human type III collagen has better bioactivity and the effects of promoting cell migration, adhesion and proliferation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a map of the recombinant expression vector pET30-1230 of the present invention;
FIG. 2 is a diagram showing the results of double digestion of the recombinant expression vector pET30-1230 of the present invention, wherein the first lane is Marker (15K), the second lane is recombinant expression vector pET30-1230 # 1, the third lane is recombinant expression vector # 1 digested with Nde I and Xho I, the fourth lane is recombinant expression vector # 2 pET30-1230, the fifth lane is recombinant expression vector # 2 digested with Nde I and Xho I, and the sixth lane is Marker (DL 2000).
FIG. 3 SDA-PAGE analysis of the electrophoresis pattern of the expression of human type III collagen protein, in which: 1. pre-staining a Marker with protein; 2. no.1 strain holoprotein is not induced by IPTG; 3. inducing the 1# strain holoprotein by IPTG; 4. no IPTG induction 3# strain holoprotein is not carried out; 5. inducing the 3# strain holoprotein by IPTG; 6. Blank; 7. no.1 strain supernatant was not induced with IPTG; 8. inducing the supernatant of the 1# strain by IPTG; 9. no. 3 strain supernatant was not induced with IPTG; 10. inducing the supernatant of the 3# strain by IPTG; 11. the protein is pre-stained with Marker.
FIG. 4 is a graph showing the results of recombinant human type III collagen identification, in which: m is Marker, 1, inducing 1# strain holoprotein by IPTG; 2. inducing the 1# strain holoprotein by IPTG; 3. no IPTG induction 3# strain holoprotein is not carried out; 4. inducing the 3# strain holoprotein by IPTG; 5. blank; 6. no.1 strain supernatant was not induced with IPTG; 7. inducing the supernatant of the 1# strain by IPTG; 8. no. 3 strain supernatant was not induced with IPTG; 9. the supernatant of the 3# strain was induced with IPTG.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail and fully with reference to the following embodiments.
The sources of reagents and materials in the examples of the invention are as follows:
the plasmid PET30a (+) was supplied by Novagen;
the DNA gel recovery kit is provided by Biotechnology engineering (Shanghai) Co., Ltd;
the plasmid rapid extraction kit is provided by biological engineering (Shanghai) Co.
Example 1 Gene design and Synthesis
(1) Gene design: based on the amino acid sequence of human type III collagen (UniProtKB/Swiss-Prot: P02461.4), SEQ ID NO: 1:
MYDSYDVKSGVAVGGLAGYPGPPGPPPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESPGGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGVGAAAIAGIGGEKAGGFAPYYHHHHHH
the gene Sequence is reversely designed by using an online design tool Jcat (http:// www.jcat.de /), NdeI and XhoI enzyme cutting sites are removed in the design process aiming at the preferable codon required by the expression of host Escherichia coli, the later gene operation is facilitated, the optimized gene Sequence is shown as SEQ ID NO:2, and compared with the original gene Sequence (NCBI Reference Sequence: NM-000090.3): the rare codon of the escherichia coli is reduced from 29 wild type codons to 1, which is beneficial to the efficient expression of the gene by the escherichia coli of a host bacterium.
The optimized gene sequence is shown as SEQ ID NO:
ATGTACGACTCTTACGACGTTAAATCTGGTGTTGCTGTTGGTGGTCTGGCTGGTTACCCGGGTCCGCCGGGTCCGCCGCCGGGTCCGGCTGGTCCGCCGGGTCCGCCGGGTCCGCCGGGTACCTCTGGTCACCCGGGTTCTCCGGGTTCTCCGGGTTACCAGGGTCCGCCGGGTGAACCGGGTCAGGCTGGTCCGTCTGGTCCGCCGGGTCCGCCGGGTGCTATCGGTCCGTCTGGTCCGGCTGGTAAAGACGGTGAATCTCCGGGTGGTCGTCCGGGTCGTCCGGGTGAACGTGGTCTGCCGGGTCCGCCGGGTATCAAAGGTCCGGCGGGTATACCGGGCTTCCCGGGTATGAAGGGTCACCGTGGTTTCGACGGTCGTAACGGTGAAAAAGGTGAAACCGGTGCTCCGGGTCTGAAAGGTGAAAACGGTCTGCCGGGTGAAAACGGTGCTCCGGGTCCGATGGGTCCGCGTGGTGCTCCGGGTGAACGTGGTCGTCCGGGTCTGCCGGGTGCTGCTGGTGCTCGTGGTAACGACGGTGCTCGTGGTTCTGACGGTCAGCCGGGTCCGCCGGGTCCGCCAGGTACTGCTGGCTTCCCGGGTTCTCCAGGTGCTAAAGGTGAAGTTGGTCCGGCTGGTTCTCCGGGTTCTAACGGTGCTCCGGGTCAGCGTGGTGAACCGGGTCCGCAGGGTCACGCTGGTCCGCCGGGTCCGGTTGGTCCGGCTGGTAAATCTGGTGACCGTGGTGAATCTGGTCCGGCTGGTCCGGCTGGTGCTCCGGGTCCGGCTGGTTCTCGTGGTGCTCCGGGTCCGCAGGGTCCGCGTGGTGACAAAGGTGAAACCGGTGAACGTGGTGCTGCTGGTATCAAAGGTCACCGTGGTTTCCCGGGTAACCCGGGTGCTCCGGGTTCTCCGGGTCCGGCTGGTCAGCAGGGTGCTATCGGTTCTCCGGGTCCGGCTGGTCCGCGTGGTCCGGTTGGTCCGTCTGGTCCGCCGGGTAAAGACGGTACCTCTGGTCACCCGGGTCCGATCGGTCCGCCGGGTCCGCGTGGTAACCGTGGTGAACGTGGTTCTGAAGGTTCTCCGGGTCACCCGGGTCAGCCGGGTCCGCCGGGTCCGCCGGGTGCTCCGGGTCCGTGCTGCGGTGGTGTTGGTGCTGCTGCTATCGCTGGTATCGGTGGTGAAAAAGCTGGTGGTTTCGCTCCGTACTACCACCACCACCACCACCAC。
(2) gene synthesis:
the 1230 fragment, which is the gene sequence shown in SEQ ID NO.2, was submitted to Kinsley Biotechnology GmbH for total gene synthesis.
EXAMPLE 2 construction of recombinant expression vector pET30-1230
The gene SEQ ID NO.2 fragment obtained in example 1 was digested with NdeI and XhoI in the following manner: the digestion system was 50. mu.L, 5. mu.L (2. mu.g) of the 2-fragment of SEQ ID NO, 1. mu.L each of NdeI and XhoI enzymes, 5. mu.L of buffer, 50. mu.L of sterile double distilled water, and incubation at 37 ℃ for 4 hours. The plasmid PET30a (+) was digested with the same system. Obtaining linear fragments by enzyme digestion, and purifying by adopting a DNA gel recovery kitAnd (3) carrying out a ligation reaction on the target fragment by using T4 ligase, wherein the ligation system is as follows: t4 DNA ligase 1 uL, 1230 cleavage fragment 3 uL, PET30a (+) cleavage fragment 2 uL, ligation buffer 1 uL, sterilized double distilled water to make up 10 uL. Keeping the temperature at 16 ℃ for 4 h. Transforming the product after heat shock into host bacteriaE.coliDH5 alpha, spread on LB-culture resistant plates and incubated overnight at 37 ℃. Positive clones were randomly picked and cultured in LB liquid medium at 37 ℃ and 220rpm overnight. The plasmid is extracted by a plasmid rapid extraction kit, and the constructed plasmid PET30-1230 is obtained, as shown in figure 1 and figure 2.
EXAMPLE 3 construction of the engineered bacteria
Picking out single colony of Escherichia coli BL21(DE3) to inoculate in LB test tube, and culturing overnight at 37 deg.C under shaking; adding 0.5mL of overnight culture solution into a triangular flask containing 50mL of LB, and carrying out vigorous shaking culture at 37 ℃ for about 2h to enable the thalli to grow to the prophase of logarithm; transferring the bacteria into a 50mL polypropylene tube precooled by ice under an aseptic condition, and standing the tube on the ice for 10 min; centrifuging at 4 ℃ and 4000rpm, pouring out supernatant, and inverting the tube to enable residual liquid to flow out as much as possible; 6mL of 0.1mol/L CaCl precooled with ice are added2Resuspending the pellet, and placing on ice for 30 min; centrifuging at 4 deg.C and 3000rpm, pouring out supernatant, and inverting the tube to allow the residual liquid to flow out as much as possible; 1.2mL of 0.1mol/L CaCl precooled with ice was added2Resuspending the precipitate (if preparing competent cells to be preserved at-70 ℃, adding 0.1mol/L CaCl2 suspension thallus containing 20% glycerol), standing at 4 ℃ for 5-24 h, and transforming; aspirate 200. mu.L of competent cell suspension and add DNA (volume)<10μL, DNA<50 ng) and mixing gently, and standing on ice for 30 min; standing in 42 deg.C water bath, heat-shocking for 90s, immediately cooling on ice; adding 500 μ L liquid LB culture solution, mixing, placing into 37 deg.C shaking table, shaking at low speed for recovery for 45min (or directly placing into 37 deg.C water bath for recovery for 1h after adding LB, and shaking the tube to suspend the cells); the transformed cells were pipetted and spread on a plate containing antibiotics, and placed in a 37 ℃ incubator for inverted culture, and the grown colony was the transformant PET30-1230/BL21(DE 3).
Example 4 inducible expression of the engineered bacteria
A single colony is picked up in LB liquid culture medium containing 50 mu g/mL Kan, cultured at 37 ℃ and 200rpm overnight to become activated seeds, then inoculated into M9 culture medium with the inoculum size of 2 percent, cultured at 23 ℃ and 200rpm for 14h, added with IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration of 0.05mM for induction expression, cultured for 12h continuously, and centrifuged to collect thalli.
The preparation method of the M9 culture medium comprises the following steps:
(1) preparation of 1M MgSO4: MgSO4·7H2Dissolving O2.46 g in 10mL of double distilled water, and sterilizing at high pressure for later use;
(2) preparation of 1M CaCl2: CaCl2·6H2Dissolving O2.191 g in 10mL of double distilled water, and sterilizing at high pressure for later use;
(3) preparation of 5 × M9 salt solution: na (Na)2PO4·7H2O 12.8g;KH2PO4 3.0g;NaCl 0.5g;NH4Cl 1.0 g; adding 200mL of double distilled water for dissolution, and sterilizing at 121 ℃ for 15 min;
(4) preparing a 20% glucose solution: dissolving 4g of glucose in 20mL of double distilled water, and filtering and sterilizing by using a 0.22 mu M filter;
(5) sterile procedure M9 medium was prepared: 200mL of 5 XM 9 salt solution; 1M MgSO 42 mL; 20mL of 20% glucose solution; 1M CaCl20.1 mL; sterile double distilled water was added to 1000 mL.
Experimental example 1: SDS-PAGE protein gel assay
See, J. SammBruk et al, methods of the third edition of the molecular cloning guidelines.
Sample treatment: the cells prepared in example 3 were collected, added to loadingbuffer and mixed well, boiled in a water bath for 10min, and cooled naturally for use.
A Kimura SurePAGE preformed gel (4-12%) is selected for sample loading, and electrophoresis is carried out for 45-55min under 140V voltage until a bromophenol blue band runs to the bottom of the gel.
Coomassie Brilliant blue R-250 was stained using a microwave oven: 1) preparing a dyeing solution: coomassie Brilliant blue R250 was dissolved in 40% ethanol and 10% acetic acid to a final concentration of 0.1% (W/V). 2) Preparing a decoloring solution: the final concentration of 10% (V/V) ethanol and 7.5% (V/V) acetic acid were dissolved together. 3) After electrophoresis, the gel plate is pried open to take out the gel, and then the gel is placed in a staining container filled with 100mL of staining solution. 4) Cover the container and put into a microwave oven to heat for 8min with high heat level. To avoid danger, care should be taken not to boil the solution. 5) Taking out the dyeing container from the microwave oven, and placing on a decolorizing shaking table to shake gently for 5min at normal temperature. 6) The staining solution was decanted and the gel carefully washed with deionized water. 7) The deionized water was decanted and 100mL of destaining solution was added. 8) Covering the cover, and heating in a microwave oven for 8min at high temperature. 9) And (4) pouring off the destaining solution, adding a new destaining solution, and repeating the step 8. 10) Taking out the mixture from the microwave oven, and placing the mixture on a decoloring shaking table to lightly shake the mixture at normal temperature until the background is clear. The results are shown in FIG. 3.
Experimental example 2 WesternBlot validation
1 protein Membrane transfer Using eBlot Rapid Wet transfer Instrument
1) Cutting 1 piece of filter paper and 1 piece of PVDF membrane with scissors, and marking one corner of the PVDF membrane with a pencil.
2) After activation of the PVDF membrane with methanol, the filter paper and PVDF membrane were soaked with transfer buffer.
3) Transfer buffer was added to the tray, and the sponge, PVDF membrane, filter paper and transfer clip were placed.
4) A piece of sponge is firstly paved on the black plate of the film transferring clamp, and then filter paper and gel are paved. After alignment, the bubbles were removed with a glass rod.
5) A small amount of transfer buffer solution is taken by a micropipette and placed on the gel, then a PVDF membrane is laid on the gel, and then filter paper and sponge are laid on the gel. After alignment, the bubbles were removed with a glass rod.
6) After the film transferring clamp is clamped and fixed, the black surface is put into a film transferring fixing device.
7) And placing the film rotating fixing device and the ice box filled with ice blocks in the film rotating groove. The transfer chamber was filled with transfer buffer.
8) The power supply is switched on, the voltage is generally constant at 100V-110V for about 1 h; the time can be properly shortened due to the small molecular weight; or constant pressure 30V, 4 ℃ film-transferring overnight; or constant current 300mA for 1h or so.
2. Blocking and antibody incubation
1) And (4) turning off the power supply, opening the membrane rotating clamp to take out the PVDF membrane, and flushing with double distilled water.
2) The PVDF membrane is placed in a sealing solution and sealed for 1h at 37 ℃ on a shaking table.
3) The blocking solution was discarded, washed with PBST buffer, incubated with primary antibody working solution, and incubated on a shaker at 37 ℃ for 1 h.
4) The primary antibody working solution was discarded, washed with PBST buffer, incubated with secondary antibody working solution, and incubated on a shaker at 37 ℃ for 1 h.
5) The secondary antibody working solution was discarded and washed with PBST buffer. The membrane was washed on a shaker for 4 times, 5min each time.
3. Development exposure
1) Residual liquid on the membrane was removed by blotting with flat paper, and the PVDF membrane was laid flat.
2) Equal volumes of liquid A and liquid B in the ECL reagent were removed by a micropipette and returned to room temperature in an EP tube.
3) Mixing, adding onto the membrane, and reacting in dark for 30-60 s.
4) The ECL mixed solution is discarded and placed in a dark box for exposure and development, and the exposure time is controlled to be about 30 s.
The results are shown in FIG. 4, and a specific band appears around 40kD after induction, which indicates that the SEQ ID NO.2 sequence fragment is specifically expressed due to induction, and the codon encoding 6 XHis fused to the 3' end is in the correct reading frame. Therefore, pET30-1230 constructed was correctly expressed, and the supernatant was substantially identical to the total protein in terms of the amount expressed, indicating that the expression product of 1230 was soluble.
It will be appreciated by those skilled in the art that the heat shock method of the present invention may be used in conjunction with scheme 25 or scheme 26 of chapter 1, scheme 25 or scheme 26, of sambrook et al, third edition of the molecular cloning, a laboratory manual; the formula of the LB culture resistant plate is shown in the chapter I of molecular cloning test guideline, third edition; the LB liquid medium formula is shown in chapter I of molecular cloning test manual, third edition, which is prior art and is not described in detail in this application.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Hubei Nake Biotech Co., Ltd
<120> recombinant human III type collagen and prokaryotic expression method thereof
<130> 1
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 410
<212> PRT
<213> type III collagen
<400> 1
Met Tyr Asp Ser Tyr Asp Val Lys Ser Gly Val Ala Val Gly Gly Leu
1 5 10 15
Ala Gly Tyr Pro Gly Pro Pro Gly Pro Pro Pro Gly Pro Ala Gly Pro
20 25 30
Pro Gly Pro Pro Gly Pro Pro Gly Thr Ser Gly His Pro Gly Ser Pro
35 40 45
Gly Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln Ala Gly
50 55 60
Pro Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser Gly Pro
65 70 75 80
Ala Gly Lys Asp Gly Glu Ser Pro Gly Gly Arg Pro Gly Arg Pro Gly
85 90 95
Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gly Ile
100 105 110
Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn
115 120 125
Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn Gly
130 135 140
Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala
145 150 155 160
Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg
165 170 175
Gly Asn Asp Gly Ala Arg Gly Ser Asp Gly Gln Pro Gly Pro Pro Gly
180 185 190
Pro Pro Gly Thr Ala Gly Phe Pro Gly Ser Pro Gly Ala Lys Gly Glu
195 200 205
Val Gly Pro Ala Gly Ser Pro Gly Ser Asn Gly Ala Pro Gly Gln Arg
210 215 220
Gly Glu Pro Gly Pro Gln Gly His Ala Gly Pro Pro Gly Pro Val Gly
225 230 235 240
Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Ser Gly Pro Ala Gly Pro
245 250 255
Ala Gly Ala Pro Gly Pro Ala Gly Ser Arg Gly Ala Pro Gly Pro Gln
260 265 270
Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu Arg Gly Ala Ala Gly
275 280 285
Ile Lys Gly His Arg Gly Phe Pro Gly Asn Pro Gly Ala Pro Gly Ser
290 295 300
Pro Gly Pro Ala Gly Gln Gln Gly Ala Ile Gly Ser Pro Gly Pro Ala
305 310 315 320
Gly Pro Arg Gly Pro Val Gly Pro Ser Gly Pro Pro Gly Lys Asp Gly
325 330 335
Thr Ser Gly His Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Asn
340 345 350
Arg Gly Glu Arg Gly Ser Glu Gly Ser Pro Gly His Pro Gly Gln Pro
355 360 365
Gly Pro Pro Gly Pro Pro Gly Ala Pro Gly Pro Cys Cys Gly Gly Val
370 375 380
Gly Ala Ala Ala Ile Ala Gly Ile Gly Gly Glu Lys Ala Gly Gly Phe
385 390 395 400
Ala Pro Tyr Tyr His His His His His His
405 410
<210> 2
<211> 1230
<212> DNA
<213> optimized Gene sequence (1230)
<400> 2
atgtacgact cttacgacgt taaatctggt gttgctgttg gtggtctggc tggttacccg 60
ggtccgccgg gtccgccgcc gggtccggct ggtccgccgg gtccgccggg tccgccgggt 120
acctctggtc acccgggttc tccgggttct ccgggttacc agggtccgcc gggtgaaccg 180
ggtcaggctg gtccgtctgg tccgccgggt ccgccgggtg ctatcggtcc gtctggtccg 240
gctggtaaag acggtgaatc tccgggtggt cgtccgggtc gtccgggtga acgtggtctg 300
ccgggtccgc cgggtatcaa aggtccggcg ggtataccgg gcttcccggg tatgaagggt 360
caccgtggtt tcgacggtcg taacggtgaa aaaggtgaaa ccggtgctcc gggtctgaaa 420
ggtgaaaacg gtctgccggg tgaaaacggt gctccgggtc cgatgggtcc gcgtggtgct 480
ccgggtgaac gtggtcgtcc gggtctgccg ggtgctgctg gtgctcgtgg taacgacggt 540
gctcgtggtt ctgacggtca gccgggtccg ccgggtccgc caggtactgc tggcttcccg 600
ggttctccag gtgctaaagg tgaagttggt ccggctggtt ctccgggttc taacggtgct 660
ccgggtcagc gtggtgaacc gggtccgcag ggtcacgctg gtccgccggg tccggttggt 720
ccggctggta aatctggtga ccgtggtgaa tctggtccgg ctggtccggc tggtgctccg 780
ggtccggctg gttctcgtgg tgctccgggt ccgcagggtc cgcgtggtga caaaggtgaa 840
accggtgaac gtggtgctgc tggtatcaaa ggtcaccgtg gtttcccggg taacccgggt 900
gctccgggtt ctccgggtcc ggctggtcag cagggtgcta tcggttctcc gggtccggct 960
ggtccgcgtg gtccggttgg tccgtctggt ccgccgggta aagacggtac ctctggtcac 1020
ccgggtccga tcggtccgcc gggtccgcgt ggtaaccgtg gtgaacgtgg ttctgaaggt 1080
tctccgggtc acccgggtca gccgggtccg ccgggtccgc cgggtgctcc gggtccgtgc 1140
tgcggtggtg ttggtgctgc tgctatcgct ggtatcggtg gtgaaaaagc tggtggtttc 1200
gctccgtact accaccacca ccaccaccac 1230
Claims (6)
1. A recombinant human type III collagen is characterized in that the amino acid sequence is shown as SEQ ID NO 1.
2. The encoding gene of human type III collagen of claim 1, wherein the nucleotide sequence of the encoding gene is represented by SEQ ID NO. 2.
3. A recombinant expression plasmid pET30-1230 comprising the gene encoding the human type III collagen of claim 2.
4. The recombinant expression plasmid pET30-1230 according to claim 3, wherein the gene encoding human type III collagen is inserted between the NdeI and XhoI cleavage sites of the expression vector PET30a (+).
5. A recombinant genetically engineered bacterium containing the recombinant expression vector pET30-1230 according to claim 3 or 4, wherein the host cell of the recombinant genetically engineered bacterium is Escherichia coli BL21(DE 3).
6. The human type III collagen prokaryotic expression method according to claim 1, characterized by comprising the following steps:
(1) gene design and synthesis: reversely designing a gene sequence according to the amino acid sequence SEQ ID NO:1 of the human type III collagen, carrying out codon optimization to obtain a gene sequence SEQ ID NO:2, and carrying out whole-gene synthesis according to the gene sequence shown in SEQ ID NO: 2;
(2) construction of recombinant expression vector pET 30-1230: the encoding gene SEQ ID NO 2 of the human type III collagen is inserted between enzyme cutting sites NdeI and XhoI of an expression vector pET30a (+), and is transformed into host bacteria by a heat shock methodE .coliIn DH 5. alpha. positive clones were selected and usedExtracting plasmids by using the plasmid rapid extraction kit to obtain a recombinant expression vector pET 30-1230;
(3) construction of transformant BL21(DE3)/pET 30-1230: transforming the recombinant plasmid pET30-1230 into a competent cell BL21(DE3) through heat shock, and screening to obtain recombinant gene engineering bacteria;
(4) inducing expression: inoculating the obtained recombinant gene engineering bacteria into an M9 culture medium, culturing overnight at 37 ℃, inoculating the obtained recombinant gene engineering bacteria into an M9 culture medium with the inoculation amount of 2%, culturing for 14h at 23 ℃, adding IPTG (0.05 mM) for induction expression, continuously culturing for 12h, and centrifugally collecting thalli.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911384510.2A CN111087463B (en) | 2019-12-28 | 2019-12-28 | Recombinant human III-type collagen and prokaryotic expression method thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911384510.2A CN111087463B (en) | 2019-12-28 | 2019-12-28 | Recombinant human III-type collagen and prokaryotic expression method thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN111087463A CN111087463A (en) | 2020-05-01 |
CN111087463B true CN111087463B (en) | 2021-02-19 |
Family
ID=70398383
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201911384510.2A Active CN111087463B (en) | 2019-12-28 | 2019-12-28 | Recombinant human III-type collagen and prokaryotic expression method thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111087463B (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111087464A (en) * | 2019-12-28 | 2020-05-01 | 河北纳科生物科技有限公司 | Recombinant human III-type collagen with functional structure and expression method thereof |
Families Citing this family (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111793127B (en) * | 2020-06-17 | 2021-11-02 | 江南大学 | III type human collagen, encoding gene, recombinant bacterium and application thereof |
CN113880941B (en) * | 2020-07-03 | 2023-11-28 | 江苏江山聚源生物技术有限公司 | Recombinant humanized IxIII collagen, expression strain and application thereof |
CN112851797B (en) * | 2020-11-27 | 2021-10-01 | 河北纳科生物科技有限公司 | Recombinant human III-type collagen and preparation method and application thereof |
CN112626074B (en) * | 2021-01-11 | 2021-11-23 | 肽源(广州)生物科技有限公司 | Hydroxyproline-modified recombinant human III-type collagen mature peptide and preparation method and application thereof |
CN112980865A (en) * | 2021-03-22 | 2021-06-18 | 可孚医疗科技股份有限公司 | Construction method of recombinant human-like collagen engineering bacteria |
CN112941081B (en) * | 2021-04-16 | 2023-04-21 | 广州启点生物科技有限公司 | Fibronectin mutant and preparation method and application thereof |
CN114671946B (en) * | 2021-12-24 | 2022-11-01 | 河北纳科生物科技有限公司 | Recombinant human III-type collagen and preparation method and application thereof |
CN114539390B (en) * | 2022-03-02 | 2023-07-18 | 广州美神生物科技有限公司 | Recombinant III type humanized collagen C3, expression vector, expression strain, expression method and application thereof |
CN114853881B (en) * | 2022-05-24 | 2023-06-16 | 华南理工大学 | Recombinant humanized fusion collagen and efficient hydroxylation method and application thereof |
CN116813749B (en) * | 2023-06-13 | 2024-01-30 | 广州启点生物科技有限公司 | Recombinant humanized III type collagen and preparation method and application thereof |
CN117126263A (en) * | 2023-08-31 | 2023-11-28 | 上海曙雅生物科技有限公司 | Recombinant human-derived III-type collagen and preparation method and application thereof |
CN117304306B (en) * | 2023-09-28 | 2024-06-04 | 广东普言生物科技有限公司 | Recombinant III type collagen Pro.C3 and preparation method and application thereof |
CN117986355B (en) * | 2024-04-03 | 2024-06-11 | 江苏亨瑞生物医药科技有限公司 | Recombinant humanized III type collagen and preparation method and application thereof |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110194795A (en) * | 2019-06-13 | 2019-09-03 | 郭伟 | A kind of recombination human source collagen and its application |
-
2019
- 2019-12-28 CN CN201911384510.2A patent/CN111087463B/en active Active
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111087464A (en) * | 2019-12-28 | 2020-05-01 | 河北纳科生物科技有限公司 | Recombinant human III-type collagen with functional structure and expression method thereof |
Also Published As
Publication number | Publication date |
---|---|
CN111087463A (en) | 2020-05-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111087463B (en) | Recombinant human III-type collagen and prokaryotic expression method thereof | |
CN111087464B (en) | Recombinant human III-type collagen with functional structure and expression method thereof | |
CN113621052B (en) | Recombinant I-type humanized collagen polypeptide and preparation method and application thereof | |
CN106554410B (en) | A kind of recombination human source collagen and its encoding gene and preparation method | |
CN109575126B (en) | Polypeptides, method for the production and use thereof | |
CN112851797B (en) | Recombinant human III-type collagen and preparation method and application thereof | |
CN114805551B (en) | Recombinant type III collagen and preparation method thereof | |
CN102146135A (en) | Recombinant human-like collagen and production method thereof | |
CN1793177A (en) | Recombined collagen and synthesizing and expressing purifying process thereof | |
CN104402975B (en) | Anti-aging small peptide and preparation method thereof | |
CN112980865A (en) | Construction method of recombinant human-like collagen engineering bacteria | |
CN114853881A (en) | Recombinant human fusion collagen and efficient hydroxylation method and application thereof | |
CN118047857A (en) | Preparation method of biological synthetic human body structural material | |
CN114957449A (en) | Preparation method of recombinant human type III collagen | |
CN106046151A (en) | Recombinant human soluble high-activity collagen type I and preparation method thereof | |
CN103193887B (en) | Recombinant porcine IL2-Fc (interteukin-2-Fc) fusion protein as well as encoding gene and expressing method of fusion protein | |
CN113880941B (en) | Recombinant humanized IxIII collagen, expression strain and application thereof | |
TWI751154B (en) | A preparation method of recombinant human granulocyte colony stimulating factor | |
CN111363028B (en) | Recombinant human type I collagen, expression strain and construction method thereof | |
CN105254745B (en) | A method of producing pig source adiponectin | |
CN1865286A (en) | Double function epidermal growth factor and its preparation method and uses | |
CN114671946B (en) | Recombinant human III-type collagen and preparation method and application thereof | |
CN103012578B (en) | Recombinant porcine interleukin 2, and encoding gene and expression method thereof | |
CN1803846A (en) | Hybrid protein of p53 protein epitope(SQAMDDLMLS) and filobactivirus gene 8 protein and application thereof | |
CN105859892A (en) | Collagen-like protein-human basic fibroblast growth factor fusion protein and preparation method thereof |
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 | ||
TR01 | Transfer of patent right | ||
TR01 | Transfer of patent right |
Effective date of registration: 20220509 Address after: 050035 room 2208, 22 / F, block a, Huigu building, No. 95 Qilian street, high tech Zone, Shijiazhuang, Hebei Patentee after: Hebei NACO Biotechnology Co.,Ltd. Patentee after: Shandong Furuida biology Co., Ltd Address before: 050035 2208, 22 / F, block a, Huigu building, 95 Qilian street, high tech Zone, Shijiazhuang City, Hebei Province Patentee before: Hebei NACO Biotechnology Co.,Ltd. |