CN116497008B - Matrix metalloproteinase 8 mutant MMP8-P326V and encoding gene and application thereof - Google Patents
Matrix metalloproteinase 8 mutant MMP8-P326V and encoding gene and application thereof Download PDFInfo
- Publication number
- CN116497008B CN116497008B CN202310449149.7A CN202310449149A CN116497008B CN 116497008 B CN116497008 B CN 116497008B CN 202310449149 A CN202310449149 A CN 202310449149A CN 116497008 B CN116497008 B CN 116497008B
- Authority
- CN
- China
- Prior art keywords
- mmp8
- matrix metalloproteinase
- mutant
- recombinant
- wound healing
- 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
- 102000056189 Neutrophil collagenases Human genes 0.000 title claims abstract description 59
- 108030001564 Neutrophil collagenases Proteins 0.000 title claims abstract description 59
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 44
- 101000990908 Homo sapiens Neutrophil collagenase Proteins 0.000 claims abstract description 38
- 102100030411 Neutrophil collagenase Human genes 0.000 claims abstract description 32
- 230000029663 wound healing Effects 0.000 claims abstract description 26
- 102000002274 Matrix Metalloproteinases Human genes 0.000 claims abstract description 18
- 108010000684 Matrix Metalloproteinases Proteins 0.000 claims abstract description 18
- 102000008186 Collagen Human genes 0.000 claims abstract description 16
- 108010035532 Collagen Proteins 0.000 claims abstract description 16
- 229920001436 collagen Polymers 0.000 claims abstract description 16
- 239000000835 fiber Substances 0.000 claims abstract description 15
- 206010061218 Inflammation Diseases 0.000 claims abstract description 14
- 230000004054 inflammatory process Effects 0.000 claims abstract description 14
- 230000008021 deposition Effects 0.000 claims abstract description 10
- 206010072170 Skin wound Diseases 0.000 claims abstract description 9
- 230000035876 healing Effects 0.000 claims abstract description 9
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 37
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 37
- 230000001737 promoting effect Effects 0.000 claims description 15
- 102000004169 proteins and genes Human genes 0.000 claims description 14
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 6
- 230000002401 inhibitory effect Effects 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000003776 cleavage reaction Methods 0.000 claims 1
- 108010030074 endodeoxyribonuclease MluI Proteins 0.000 claims 1
- 230000007017 scission Effects 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 17
- 241000282414 Homo sapiens Species 0.000 abstract description 10
- 230000008439 repair process Effects 0.000 abstract description 4
- 230000004089 microcirculation Effects 0.000 abstract description 3
- 230000019612 pigmentation Effects 0.000 abstract description 3
- 231100000241 scar Toxicity 0.000 abstract description 3
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 35
- 208000027418 Wounds and injury Diseases 0.000 description 34
- 206010052428 Wound Diseases 0.000 description 31
- 241000700159 Rattus Species 0.000 description 17
- 239000013612 plasmid Substances 0.000 description 17
- 238000002474 experimental method Methods 0.000 description 15
- 230000035772 mutation Effects 0.000 description 15
- 238000013508 migration Methods 0.000 description 13
- 230000005012 migration Effects 0.000 description 11
- 239000002609 medium Substances 0.000 description 9
- 210000003491 skin Anatomy 0.000 description 9
- 241001481833 Coryphaena hippurus Species 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 238000010186 staining Methods 0.000 description 8
- 230000035755 proliferation Effects 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- ZQFGRJWRSLZCSQ-ZSFNYQMMSA-N verteporfin Chemical group C=1C([C@@]2([C@H](C(=O)OC)C(=CC=C22)C(=O)OC)C)=NC2=CC(C(=C2C=C)C)=NC2=CC(C(=C2CCC(O)=O)C)=NC2=CC2=NC=1C(C)=C2CCC(=O)OC ZQFGRJWRSLZCSQ-ZSFNYQMMSA-N 0.000 description 7
- 229960003895 verteporfin Drugs 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 230000012292 cell migration Effects 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 210000002510 keratinocyte Anatomy 0.000 description 6
- 230000037314 wound repair Effects 0.000 description 6
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 5
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 5
- 210000002744 extracellular matrix Anatomy 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 241000282412 Homo Species 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 208000002109 Argyria Diseases 0.000 description 3
- 102000000905 Cadherin Human genes 0.000 description 3
- 108050007957 Cadherin Proteins 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- 241001125840 Coryphaenidae Species 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000005741 Metalloproteases Human genes 0.000 description 3
- 108010006035 Metalloproteases Proteins 0.000 description 3
- 208000028990 Skin injury Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 3
- 229960005091 chloramphenicol Drugs 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 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 3
- 239000012634 fragment Substances 0.000 description 3
- 210000004907 gland Anatomy 0.000 description 3
- 210000003780 hair follicle Anatomy 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 208000014674 injury Diseases 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 241000283153 Cetacea Species 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 206010063560 Excessive granulation tissue Diseases 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 208000012641 Pigmentation disease Diseases 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000003044 adaptive effect Effects 0.000 description 2
- 230000033115 angiogenesis Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 210000001126 granulation tissue Anatomy 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000004850 protein–protein interaction Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 238000007634 remodeling Methods 0.000 description 2
- 206010040882 skin lesion Diseases 0.000 description 2
- 231100000444 skin lesion Toxicity 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 102000036664 ADAM10 Human genes 0.000 description 1
- 108091007504 ADAM10 Proteins 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000019300 CLIPPERS Diseases 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 108050009160 DNA polymerase 1 Proteins 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 241000305491 Gastrodia elata Species 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 208000002260 Keloid Diseases 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 241000282577 Pan troglodytes Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 101150076211 TH gene Proteins 0.000 description 1
- 102100031873 Transcriptional coactivator YAP1 Human genes 0.000 description 1
- 101710193680 Transcriptional coactivator YAP1 Proteins 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 206010048629 Wound secretion Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- INJRKJPEYSAMPD-UHFFFAOYSA-N aluminum;silicic acid;hydrate Chemical compound O.[Al].[Al].O[Si](O)(O)O INJRKJPEYSAMPD-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 208000021930 chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids Diseases 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000002498 deadly effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 210000004207 dermis Anatomy 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 210000003195 fascia Anatomy 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000001117 keloid Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000037311 normal skin Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 230000003334 potential effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 108020001775 protein parts Proteins 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 210000000434 stratum corneum Anatomy 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6489—Metalloendopeptidases (3.4.24)
- C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
-
- 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]
-
- 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
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/24—Metalloendopeptidases (3.4.24)
- C12Y304/24034—Neutrophil collagenase (3.4.24.34)
-
- 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
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Dermatology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a matrix metalloproteinase 8 mutant MMP8-P326V and a coding gene and application thereof. The invention discloses a matrix metalloproteinase 8 mutant MMP8-P326V, which is formed by mutating Pro at 326 th position of wild matrix metalloproteinase 8 (MMP 8) shown in SEQ ID NO.1 into Val. The invention further discloses application of the matrix metalloproteinase 8 mutant MMP8-P326V in wound healing. Compared with wild MMP8, the matrix metalloproteinase 8 mutant MMP8-P326V has better anti-inflammatory, detumescence and skin repair effects; can improve microcirculation; the side effects of pigmentation, collagen fiber deposition and scar generation caused by wild MMP8 are relieved to a great extent; has great application value. The mutant disclosed by the invention promotes wound healing to be stronger than MMP8, can inhibit skin healing negative events such as excessive collagen fiber deposition, excessive inflammation and the like caused by MMP8, and provides a new direction and thought for related research on improving the human skin wound healing capability.
Description
Technical Field
The invention relates to the technical field of molecular biology. More particularly, it relates to a matrix metalloproteinase 8 mutant MMP8-P326V and its coding gene and application.
Background
Matrix Metalloproteinases (MMPs) belong to a class of highly conserved endogenous proteases in the ECM hydrolase class, the activity of which is highly dependent on Ca 2+ And Zn contained therein 2+ The active centers of such proteases act together by binding metal ions to form a complex with surrounding water molecules, catalyzing and hydrolyzing proteins. MMPs are produced in the form of zymogens of MMPs without enzyme activity and secreted extracellularly, involved in the physiological and biochemical processes of organisms.
MMP8 belongs to a member of the MMPs family, has the ability to degrade the extracellular matrix, and plays an important role in wound healing. MMP8 can promote the maturation of metalloproteinase domain protein 10 (ADAM 10), and then increase the shedding of the nitrogen end of E-cadherin (E-cadherin) on vascular wall stem cells, form soluble E-cadherin, and promote angiogenesis; MMP8 can also accelerate remodeling of extracellular matrix by degrading collagen fibers [1] Improving cell migration ability [2] Promoting regeneration of skin wound tissue. MMP8 addition has the effect of accelerating the healing of diabetic wounds. However, excessive accumulation of MMP8 can lead to excessive collagen synthesis [3] To the progression of fibrosis [4] Even keloids; can also cause abnormal degradation of collagen I, II and III and delay wound healing [5] And even cause ulcers to form.
Primary structural element of MMP8 proteinParts, including signal peptide, propeptide domains, catalytic regions, hinge region domains, and heme-like domains. The functions of these domains are different depending on the structure and sequence, and may be different from each other, and may be combined to form a spatial complex. The C-terminal of MMP8 has a heme-like domain which determines the binding mode of substrate specificity, is an important structure when degrading collagen with triple helix structure, and can specifically identify the binding region of endogenous inhibitor so as to play an important role. The 326 th site of MMP8 is in the heme-like domain of the protein, which is linked to the heme-like C-terminus of the catalytic domain via a flexible hinge region, is important for localization, protein-protein interactions and substrate specificity, and is precisely the critical site for MMP8 interactions with metalloproteinase Tissue Inhibitors (TIMPs) [6] . This site has undergone a specific mutation in dolphin with an abnormally strong wound healing capacity (Pro→Val, P326V), and it is predicted that the P326V mutation may not only affect MMP8 function but also cause a change in its protein structure. Therefore, the novel matrix metalloproteinase 8 mutant obtained by site-directed mutagenesis may have important significance in improving skin wound healing.
Reference is made to:
[1]Lurier EB,Dalton D,Dampier W,et al.Transcriptome analysis of IL-10-stimulated(M2c)macrophagesbynext-generation sequencing[J].Immunobiology,2017,222(7):847-856.
[2]Xiao Q,Zhang F,Grassia G,et al.Matrix metalloproteinase-8promotes vascular smooth muscle cell proliferation and neointima formation[J].Arterioscler Thromb Vasc Biol,2014,34(1):90-98.
[3]Sidgwick GP,Bayat A.Extracellular matrix molecules implicated in hypertrophic and keloid scarring[J].JEurAcadDermatol Venereol,2012,26(2):141-152.
[4]Stallmach A,SchuppanD,Riese HH,et al.Increased collagen type III synthesis by fibroblasts isolated from strictures ofpatients withCrohn's disease[J].Gastroenterology,1992,102(6):1920-1929.
[5]Stone RC,Stojadinovic O,Sawaya AP,et al.A bioengineered living cell construct activates metallothionein/zinc/MMP8 and inhibits TGFβto stimulate remodeling of fibrotic venous leg ulcers[J].Wound Repair Regen,2020,28(2):164-176.
[6]Bassiouni W,Ali MAM,Schulz R.Multifunctional intracellular matrix metalloproteinases:implications in disease[J].Febsj,2021,288(24):7162-7182.
[7]Zasloff M.Observations on the remarkable(and mysterious)wound-healing process of the bottlenose dolphin[J].J InvestDermatol,2011,131(12):2503-2505.
[8]Dwyer SL,Kozmian-Ledward L,Stockin KA.Short-term survival of severe propeller strike injuries and observations onwoundprogressioninabottlenose dolphin[J].
[9]Su CY,Hughes MW,Liu TY,et al.Defining Wound Healing Progression in Cetacean Skin:Characteristics ofFull-Thickness Wound Healing in Fraser's Dolphins(Lagenodelphis hosei)[J].Animals(Basel),2022,12(5).
[10]de Oliveira RC,Wilson SE.Fibrocytes,Wound Healing,and Corneal Fibrosis[J].Invest OphthalmolVis Sci,2020,61(2):28.
[11]Martin P,Nunan R.Cellular and molecular mechanisms ofrepair in acute and chronic wound healing[J].BrJDermatol,2015,173(2):370-378.
[12]Kim SY,NairMG.Macrophages inwoundhealing:activation andplasticity[J].Immunol Cell Biol,2019,97(3):258-267.
disclosure of Invention
The invention aims to provide a key protein for promoting skin wound healing, a mutant MMP8-P326V of matrix metalloproteinase 8, and a coding gene and application thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention firstly provides a matrix metalloproteinase 8 mutant MMP8-P326V, wherein the matrix metalloproteinase 8 mutant MMP8-P326V is obtained by mutating Pro at 326 th site of wild matrix metalloproteinase 8MMP8 (sequence shown as SEQ ID NO. 1) shown as NM_002424 in NCBI database transcripts into Val.
Further, the amino acid sequence of the matrix metalloproteinase 8 mutant MMP8-P326V is a protein shown as SEQ ID NO. 2.
The invention further provides a coding gene of the mutant MMP8-P326V of the matrix metalloproteinase 8. The nucleotide sequence of the coding gene of the mutant MMP8-P326V of the matrix metalloproteinase 8 is shown as SEQ ID NO. 3. It should be noted that, since the same amino acid may have a plurality of different codons to determine, the encoding gene of the mutant MMP8-P326V of the matrix metalloproteinase 8 may also be a gene nucleotide sequence obtained by mutating one or more nucleotides of the encoding gene of the wild matrix metalloproteinase 8 shown in SEQ ID NO.4 to form synonymous mutation, which can also encode the mutant MMP8-P326V of the matrix metalloproteinase 8 of the present invention.
The invention protects a recombinant vector containing the coding gene of the matrix metalloproteinase 8 mutant MMP8-P326V.
Further, the recombinant vector is obtained by mutating Pro of 326 th gene of the encoding gene of the matrix metalloproteinase 8 into Val and keeping other sequences of pLenti-C-mGFP-P2A-Puro unchanged, and is a recombinant vector pLenti-C-mGFP-P2A-Puro-MMP8-P326V.
The invention further provides a preparation method of the matrix metalloproteinase 8 mutant MMP8-P326V, which comprises the following steps:
(1) Designing a primer carrying a mutation site aiming at the mutation site of a matrix metalloproteinase 8 mutant MMP8-P326V, and carrying out site-directed mutation PCR by taking a plasmid containing a coding gene of a wild matrix metalloproteinase 8 as a template to obtain a recombinant vector containing the coding gene of the matrix metalloproteinase 8 mutant MMP8-P326V;
(2) Transforming host cells by the recombinant vector to obtain recombinant bacteria;
(3) Culturing recombinant bacteria and inducing expression protein;
(4) Recovering and purifying to obtain the matrix metalloproteinase 8 mutant MMP8-P326V.
Further, the sequences of the primers are shown as SEQ ID NO.7 and SEQ ID NO. 8.
The invention also protects recombinant proteins comprising the recombinant vector of the matrix metalloproteinase 8 mutant MMP8-P326V.
According to the invention, the proliferation and migration conditions of immortalized human keratinocytes are characterized by scratch and Transwell experiments, which shows that the rh-mut-MMP8 has stronger proliferation and migration promoting capacity (P < 0.005).
Further, haCaT was suggested by cell scratch and Transwell experiments rh-mut The cells have stronger cell activity and migration capability. This suggests that rh-mut-MMP8 may have a better promoting effect on wound healing than rh-MMP8, indicating the effect of the recombinant protein matrix metalloproteinase 8 mutant MMP8-P326V on SD rat skin repair.
The invention also protects application of the recombinant protein matrix metalloproteinase 8 mutant MMP8-P326V in preparing products for promoting skin wound healing and inhibiting skin healing negative events such as excessive collagen fiber deposition (Masson and H & E staining experiments) and excessive inflammation (wound repair condition of SD rat full-layer skin injury model) caused by MMP8.
The invention discovers that the rh-mut-MMP8 has the same effect as the verteporfin group in terms of wound healing promoting effect, wound smoothness after wound recovery and wound color, has better effect than the rh-wt-MMP8, and shows that the recombinant protein matrix metalloproteinase 8 mutant MMP8-P326V has good skin injury repairing effect on rats. Experiments also indicate that rh-mut-MMP8 can inhibit the progress of inflammation, and inhibit excessive collagen fiber deposition and excessive inflammation caused by recombinant protein MMP8, thereby having good function of promoting skin wound healing. Compared with the wild MMP8 of the recombinant protein, the MMP8 mutant of the recombinant protein matrix metalloproteinase 8 has better anti-inflammatory, detumescence and skin repair effects; can improve microcirculation; the side effects of pigmentation, collagen fiber deposition and scar generation caused by wild MMP8 are relieved to a great extent.
Compared with the prior art, the invention has the following beneficial effects:
the invention obtains a matrix metalloproteinase 8 mutant MMP8-P326V by changing Pro at 326 th site of wild matrix metalloproteinase 8MMP8 into Val for the first time, and discloses a preparation method of the mutant, and the recombinant protein matrix metalloproteinase 8 mutant MMP8-P326V inhibits excessive deposition and excessive inflammation of collagen fibers caused by recombinant protein MMP8 and has good function of promoting skin wound healing. The recombinant protein matrix metalloproteinase 8 mutant MMP8-P326V has the effects of resisting inflammation, detumescence, promoting skin healing, improving microcirculation, inhibiting pigmentation, inhibiting collagen fiber deposition and reducing scar generation, and has great application value.
Drawings
FIG. 1 is a diagram of key information for screening out MMP8 genes by adaptive evolution analysis; A. species trees for adaptive evolution analysis, grey is aquatic organism. And predicting positive selection signals of the D pivot and the W pivot by using a pivot model. B. Wien diagram, gene a is a wound healing and infection gene. Gene B is a unique NSGs (negative selection gene) of dolphin. Gene C is a PSGs (positive selection gene) unique to dolphin. C. A gene interaction network map of specific PSGs of dolphin and wound healing and infection genes;
FIG. 2 is an analysis of the potential effect of P326V on MMP8; A. the 326 th amino acid sequence of dolphin MMP8 gene with special wound healing capacity generates specific P-V mutation, and the sites of human, chimpanzee, mouse, goat, whale, etc. are all conserved. PolyPhen-2 predicts the functional changes due to sequence differences, suggesting that P326V may result in a functional change in MMP8, with greater probably damaging values being more likely to occur. MMP8-P326V (purple) differs from MMP8 (green) in the 3D protein domain;
FIG. 3 is a validation of matrix metalloproteinase 8 mutants; A. mutating the human MMP8 gene sequence into a sequencing result of a dolphin MMP8 gene sequence; B-D purifying the recombinant protein; coomassie brilliant blue staining, silver staining and Western blotting verification of recombinant plasmids MMP8-P326V (Mut), wild type MMP8 (Wt), vector plasmid (V) into 293T cells.
FIG. 4 is a graph of proliferation and migration of recombinant protein wild-type matrix metalloproteinase 8MMP8 and recombinant protein matrix metalloproteinase 8 mutant MMP8-P326V to human keratinocytes; after treatment of HaCaT cells with recombinant proteins rh-mut-MMP8, rh-wt-MMP8, rh-vector, respectively, proliferation and migration of the cells were examined, and control was HaCaT cells (blank control). A. Scratch experiment; B. mobility of scratch test; transwell experiments; statistical plot of transition experimental migration area (P < 0.05);
FIG. 5 is a graph comparing wound repair of a recombinant protein wild type matrix metalloproteinase 8MMP8 and a recombinant protein matrix metalloproteinase 8 mutant MMP8-P326V promoting full-thickness skin lesions of SD rats; rh-mut-MMP8, rh-wt-MMP8, rh-vector, verteporfin were used to treat SD rat full-thickness skin injury wound model, and wound repair processes of each group were recorded, optimal group: control (dilution of recombinant protein). A. Wound healing, black frame: inflammation crusting; B. closure rate of the wound; C. weight change in rats during wound repair;
FIG. 6 is a graph comparing collagen fibers of recombinant protein wild type matrix metalloproteinase 8MMP8 and recombinant protein matrix metalloproteinase 8 mutant MMP8-P326V promoting full-thickness skin lesions in SD rats; rh-mut-MMP8, rh-wt-MMP8, rh-vector, verteporfin treatment of wounds, low power hematoxylin and eosin (H & E) histological staining (left) and Masson staining (right), black boxes: hair follicle, gland, blood vessel, etc.
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments and the accompanying drawings. Like parts in the drawings are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.
EXAMPLE 1 construction of wild-type matrix metalloproteinase 8 recombinant vector (pLenti-C-mGFP-P2A-Puro-MMP 8) plasmid
Matrix metalloproteinase 8 is derived from homosapiens, the amino acid sequence of which is found in NCBI accession No.: NM-002424 (amino acid sequence shown in SEQ ID NO. 1). The gene sequence of wild matrix metalloproteinase 8 (before mutation) is subjected to colibacillus codon optimization, and gene synthesis is carried out by Kunming Tuo source trade company, so as to obtain the target gene MMP8 (the nucleotide sequence is shown as SEQ ID NO. 4).
Adding a homologous fragment of pLenti-C-mGFP-P2A-Puro plasmid at the tail end of a target gene MMP8 through PCR reaction to obtain MMP8 containing the homologous fragment;
the primers used in the PCR reaction are designed by using bioinformatics software SnapGene, and the specific sequences are as follows:
MMP8-F:CATTCTTTGGGGCTCGCTCA(SEQ ID No.5)
MMP8-R:GTGTGCTTGGTCCAGTAGGT(SEQ ID No.6)
the PCR reaction system is as follows: MMP8200ng, MMP8-F (10 uM) 4. Mu.L, MMP8-R (10 uM) 4. Mu.L, 10 XBeyoFusion TM Buffer(with Mg 2+ )5μL,dNTP mix(2.5mM)10μL,BeyoFusion TM DNA Polymerase 1. Mu.L, nucleic-Frer Water make up to 50. Mu.L.
The conditions for the PCR reaction were:
the product is transformed into E.coli HB101 competent cells to obtain a transformed bacterial solution; the transformed bacterial liquid is evenly coated on an LB screening plate (chloramphenicol concentration: 34 mug/mL), cultured overnight at 37 ℃, transformants are picked up, cultured in an LB liquid medium, plasmids are extracted, and recombinant plasmids pLenti-C-mGFP-P2A-Puro-MMP8 are obtained through enzyme digestion and PCR verification. Then the recombinant plasmid pLenti-C-mGPP 2A-Puro-MMP8 is transformed into E.coli HB101 competent cells to obtain recombinant strain E.coli HB101, which is named HB 101/pLenti-C-mGPP 2A-Puro-MMP8.
EXAMPLE 2 construction of plasmid of recombinant vector (pLenti-C-mGFP-P2A-Puro-MMP 8-P326V) for matrix metalloproteinase 8 mutant MMP8-P326V
1. Selection of mutation sites
In many mammals, dolphinsCan not only effectively control the bleeding degree after serious injury in the ocean [7] The method comprises the steps of carrying out a first treatment on the surface of the Less immediate deadly [8] The large wound which hurts the dermis layer can be healed quickly in a short time, and the damage is healed more approximate to the normal skin structure [9] . Such special healing capabilities are not available to humans. To search for its mechanism and find the differences between humans, we selected 5 species of humans, chimpanzees, mice, goats and whales with dolphins for whole genome evolution analysis (FIG. 1A). As a result, it was found that 9 genes including MMP8 are also involved in wound repair in humans, and undergo unique positive selection in dolphins (FIGS. 1B-C). And the 326 site of MMP8 is conserved in species that do not have the ability to repair specific wounds, but only mutations in dolphins (see figure 2A), which are predicted to affect not only MMP8 function (figure 2B), but also changes in its protein structure (figure 2C). The site is in a heme-like domain, exists in a C-terminal domain with a large surface, is connected to the heme-like C-terminal of the catalytic domain through a flexible hinge region, is important for localization, protein-protein interactions and substrate specificity, and site 326 is the site of MMP8 interactions with metalloproteinase Tissue Inhibitors (TIMPs) [6] . This suggests that mutation of the specific site of MMP8 in dolphin is one of the reasons that dolphin has relatively specific wound healing ability, and also one of the keys to human self-healing.
2. MMP8 mutant MMP8-P326V recombinant vector (pLenti-C-mGFP-P2A-Puro-MMP 8)
Construction of the P326V) plasmid
In order to mutate Pro at position 326 in the amino acid sequence of wild type matrix metalloproteinase 8MMP8 to Val, primers carrying mutation sites were designed with bioinformatics software SnapGene, the sequences of which are as follows:
P326V-F:ttctctattctggccatcccttgtaactggtatacaggctgctta(SEQ ID No.7)
P326V-R:aagagataagaccggtagggaacattgaccatatgtccgacgaat(SEQ ID No.8)
wherein the underlined portion represents the codon corresponding to Val at position 326 of the mutation site.
And (3) performing site-directed mutation PCR by using the primers to obtain a PCR product, and determining mutation success by sequencing (the detection result is shown in figure 3A). Then, the PCR reaction system and conditions were the same as in example 1 using the recombinant plasmid pLenti-C-mGFP-P2A-Puro-MMP8 as a template.
The PCR product was recovered, followed by addition of restriction enzyme Dpn I and incubation at 37℃for 1h to remove the template. After digestion, dpn I can be directly used for conversion or stored at-20 ℃ for standby.
The product is transformed into E.coli HB101 competent cells, the transformed bacterial liquid is evenly coated on an LB screening plate (chloramphenicol concentration: 34 mug/mL), cultured overnight at 37 ℃, the transformant is selected, cultured in an LB liquid culture medium, plasmids are extracted, and recombinant plasmids pLenti-C-mGP 2A-Puro-MMP8-P326V are obtained through enzyme digestion, PCR and sequencing verification. Then the recombinant plasmid pLenti-C-mGPFP-P2A-Puro-MMP 8-P326V is transformed into E.coli HB101 competent cells to obtain recombinant strain E.coli HB101, which is named HB 101/pLenti-C-mGPFP-P2A-Puro-MMP 8-P326V.
EXAMPLE 3 induced expression of wild type matrix metalloproteinase 8MMP8 and matrix metalloproteinase 8 mutant MMP8-P326V, isolation and purification
HB101/pLenti-C-mGFP-P2A-Puro-MMP8 obtained in example 1 and HB101/pLenti-C-mGFP-P2A-Puro-MMP8-P326V obtained in example 2 were inoculated into 3mL of LB liquid medium (chloramphenicol concentration: 34. Mu.g/mL), and cultured at 37℃for 6 hours at 180r/min for activation, and after transfer into 300mL of LB liquid medium having the same resistance, cultured at 37℃for 12 hours at 180r/min, and centrifuged at 8000r/min to collect the cells.
Re-suspending the thallus with 12mL Buffer P1, then fully cracking the thallus with 12mL Buffer P2 (NaOH and SDS), adding 12mL Buffer P3 into the crushed bacterial liquid, centrifuging for 15min with 12000r/min to remove cell fragments, and obtaining the supernatant which is the matrix metalloproteinase 8 crude enzyme liquid.
Purifying the crude enzyme solution of the matrix metalloproteinase 8 by a 0.22 mu m filter membrane to purify the matrix metalloproteinase 8: firstly, using Buffer PS with 1 column volume to balance and purify the column, then using Buffer PW to make gravity washing treatment to obtain target plasmid, adding 500uL Endo-Free Buffer EB to make elution so as to obtain the target plasmid, using 500 uL/tube to make split charging of residual liquid, quick-freezing and storing at-80 deg.C for later use.
EXAMPLE 4 wild-type matrix metalloproteinase 8MMP8 and recombinant protein matrix metalloproteinase 8 mutant MMP8-P326V induced expression and separation purification
The plasmid of interest obtained in example 3 was transfected into 293T cells, after 48h the original medium was aspirated off, both sides were rinsed with PBS, and further cultured with optimem medium for 24h, after which the medium was collected and centrifuged at 1,000rpm for 3min. The supernatant was concentrated and purified by transferring to a 100KD concentration tube (Milibo UFC 910096), and centrifuged at 4℃and 4,000rpm for 30min. After purification, 1uL of protease inhibitor was added, and the cells were lysed on ice for 30min, centrifuged at 12000g for 15min, and the protein of each cell group was extracted from the supernatant for concentration measurement. The subsequent experiments were performed after confirming no contamination with other proteins (FIGS. 3B-D) by Coomassie brilliant blue staining, silver staining and Western blotting experiments. Mut: recombinant protein matrix metalloproteinase 8 mutant MMP8-P326V; wt: recombinant protein wild type matrix metalloproteinase 8MMP8; vec: recombinant protein pLenti-C-mGFP-P2A-Puro; c: untreated groups.
EXAMPLE 5 comparison of efficiencies of recombinant protein wild-type matrix metalloproteinase 8MMP8 and recombinant protein matrix metalloproteinase 8 mutant MMP 8-P326V- -recombinant protein matrix metalloproteinase 8 mutant MMP8-P326V affects immortalized human keratinocyte scoring and Transwell experiments
The purified recombinant proteins rh-mut-MMP8 and rh-wt-MMP8 are processed under the pollution condition of no other proteins determined by purification detection such as Coomassie brilliant blue, silver staining and Western blotting experiments. To observe the effect of the recombinant protein matrix metalloproteinase 8 mutant MMP8-P326V on proliferation and migration of immortalized human keratinocytes, 2.0X10 were used 6 Immortalized human keratinocyte cell lines (HaCaT) were seeded at a density of individual cells/mL in six well plates, 2mL per well of DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin, and incubated in a cell incubator. After 24h, 11.78ug of each of rh-mut-MMP8, rh-wt-MMP8 and rh-vector was transferred into HaCaT, and after 48h, scratch and Transwell experiments were performed.
The scratch test was performed at 7X 10 5 The density of individual cells/mL, 70 ul/well, was inoculated with each set of HaCaT in six well plates with inserts added to DMEM medium. After cell attachment, the cell inserts were removed and the cells floating at the scratch were gently washed off with PBS. Healing of each group of scratches was observed with untreated HaCaT as a blank. Scratch photographs were taken at 0, 12 and 24h using an inverted microscope and width measurements were made at four points randomly selected.
The Transwell experiments were performed by placing the cells in a new 24-well plate one day in advance, adding 600mL of basal medium to each of the upper and lower chambers, and incubating the cells for 24 hours. The upper chamber was inoculated with a serum-free cell suspension at 50,000 cells per well, and the lower chamber was incubated with 10% serum medium for 24h. 600 mu L of paraformaldehyde is added into each of the upper chamber and the lower chamber, and the mixture is fixed for 10 minutes at room temperature; 600 μl of crystal violet was added to each for 10min; washing with PBS to remove excess dye, wiping off cells on the upper chamber membrane surface with a cotton swab to remove uninfected cells, observing the cells under a microscope, and counting.
The invention characterizes the proliferation and migration changes of immortalized human keratinocytes through scratch and Transwell experiments: haCaT was treated with recombinant proteins rh-mut-MMP8 and rh-wt-MMP 8. Discovery of HaCaT rh-mut Group cell scratch gap was smaller, closure efficiency (cell mobility) was higher, indicating HaCaT rh-mut Group comparison HaCaT rh-wt And HaCaT rh-v Has obviously improved cell proliferation and migration capacity (P)<0.05),HaCaT rh-wt Group, haCaT rh-v There was little difference in proliferation and migration capacity between the group and the control group (FIGS. 4A, B). This suggests HaCaT rh-mut The cells have stronger cell proliferation and migration capacity.
Further, through a Transwell experiment, haCaT was found rh-mut The number of blue-stained cells is the greatest, while HaCaT rh-wt The least number of blue-stained cells indicates HaCaT rh-mut Compared with HaCaT rh-wt And HaCaT rh-v The group significantly improved cell migration capacity (P<0.05 (fig. 4c, d), while HaCaT rh-wt And HaCaT rh-v May inhibit the migration ability of cells, suggesting that rh-mut-MMP8 may promoteInlet cell migration, rh-wt-MMP8 may play an opposite role, inhibiting migration capacity. In the process of wound repair, the improvement of the cell proliferation capacity can promote the filling of defective tissues, help to remove inflammatory substances, strengthen the cell migration capacity and promote wound closure, build a protective barrier for the organism and avoid external infection [10] . This suggests that rh-mut-MMP8 may have a better promoting effect on wound healing than rh-MMP 8.
EXAMPLE 6 Effect of the recombinant protein matrix metalloproteinase 8 mutant MMP8-P326V (pLenti-C-mGFP-P2A-Puro-MMP 8-P326V) on skin repair- -use efficacy Performance test
rh-mut-MMP8: injecting purified matrix metalloproteinase 8 mutant MMP8-P326V (pLenti-C-mGFP-P2A-Puro-MMP 8-P326V) recombinant protein;
rh-wt-MMP8: injecting purified wild-type matrix metalloproteinase 8 (pLenti-C-mGFP-P2A-Puro-MMP 8) recombinant protein;
rh-vector: injecting and purifying recombinant proteins of pLenti-C-mGFP-P2A-Puro;
verteporfin: (yes-associated protein 1 inhibitors to promote wound healing) treatment to regenerate rat wounds;
optimem: a dilution of the recombinant protein is injected.
Establishment of rat full-cortex injury model
Animals were randomly divided into 5 groups (n=3), 3 animals each weighing 180-200g SD rats, and a rat wound model was constructed.
Anesthesia was performed by intraperitoneal injection using a 1% solution of sodium pentobarbital, at a specific dose of 50mg/kg, about 0.8ml, and the specific dose varied depending on the animal body weight. Firstly, the anesthetized animals are cut off by electric hair clippers to expose skin, and after being sterilized by iodine, three equal-sized circular wound surfaces are formed on the back by using an ophthalmic trephine with the diameter of 3 cm. The wound is deep until the fascia outside the muscle is exposed. The postoperative SD rats were bred separately one cage by one cage with reference to the fully domesticated breeding conditions.
After the model is manufactured, the rats are anesthetized by the same method every other day, the wound healing condition of each group is recorded by photographing for 3 weeks, then a certain proportion is set by a ruler, the proportion is used for calculating the area in the image J, and the size setting of the picture is facilitated. After the measurement, rh-mut-MMP8, rh-wt-MMP8 and rh-vector, optimem, verteporfin were injected again.
After 21 st gastrodia elata drunk measurement record of the manufacturing model is completed, materials are obtained, healed skin tissues of the rat are cut off by using a surgical scissors, the extracted tissues are divided into two parts, one part is used for tissue staining, and the other part is used for detecting downstream genes.
The use of rh-mut-MMP8, rh-wt-MMP8, rh-vector (50. Mu.L, protein concentration in optimem buffer 20. Mu.g/mL) [11] Or control (50. Mu.L optimem buffer) to treat the wound surface locally, 1 time a day for 10d.
Animal wound models were treated with recombinant proteins rh-mut-MMP8 and rh-wt-MMP 8. Topical application to wounds at 10-fold protein levels in rats per day, with topically applied rh-mut-MMP8 accelerating wound healing of rats more significantly than the other groups (fig. 5a, b), and recombinant proteins not having a severe effect on rat body weight (fig. 5C). From the condition of wound healing in wound recovery, the rh-mut-MMP8 and verteporfin group have excellent effect of promoting wound healing on animal epidermis wound healing experiments, and the wound surface is flat and has no secretion, and the wound surface basal part is ruddy; the rh-wt-MMP8 group has small amount of secretion on the wound surface, is relatively smooth, has vigorous growth of granulation tissue, and has smooth and light red wound surface; the rh-vector group has more wound secretions, obvious bulges and black brown color; the wound surface of the optimem group has a large amount of secretion and is uneven, the granulation tissue grows slowly, and the wound surface is concave and dark gray (figure 5A). The skin wound surface has obviously reduced inflammation in the rh-mut-MMP8 group, the rh-wt-MMP8 group and the verteporfin group compared with the rh-vector group and the optimem group on 11 th day after injury, and has slight inflammation at the wound edges of the rh-vector group and the optimem group on 21 th day, while the wound surface of the rh-mut-MMP8 group is almost covered by epithelium, and no obvious inflammation is observed. It was suggested that rh-mut-MMP8 might inhibit the progression of inflammation.
Further, from HE staining, it was found that the rh-vector group and the optimem group showed massive inflammatory cell accumulation on the wound surface, and the stratum corneum fracture was substantially a bare area, with dense, linearly arranged extracellular matrix fibers, without secondary accessories (hair follicle, gland). In contrast, rh-mut-MMP8 treated wounds had higher levels of hair follicles and glands, had a lower density and random distribution of extracellular matrix fibers and neovasculature (FIG. 6), thereby significantly promoting wound re-epithelialization, angiogenesis and modulating extracellular matrix remodeling. From the results of Masson staining, the rh-wt-MMP8 group showed excessive collagen fiber deposition (severe blue staining), whereas the rh-mut-MMP8 group showed a small number of ordered collagen fiber arrangements (fig. 6).
Excessive inflammation [12] And collagen deposition are important causes of long-term non-healing wounds. Through observation of the pathological forms of the skin, the rh-mut-MMP8 can promote the degradation of collagen fibers, inhibit the progress of inflammation and promote the regeneration of the skin while accelerating the healing of wound surfaces.
Claims (6)
1. A recombinant protein matrix metalloproteinase 8 mutant MMP8-P326V is characterized in that the matrix metalloproteinase 8 mutant MMP8-P326V is obtained by mutating Pro at 326 th site of matrix metalloproteinase 8MMP8 shown in SEQ ID NO.1 into Val.
2. The matrix metalloproteinase 8 mutant MMP8-P326V of claim 1, wherein the amino acid sequence of the matrix metalloproteinase 8 mutant MMP8-P326V is a protein represented by SEQ ID No. 2.
3. The gene encoding matrix metalloproteinase 8 mutant MMP8-P326V of claim 1 or 2, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID No. 3.
4. A recombinant vector comprising the coding gene of claim 3, wherein the recombinant vector is a recombinant vector plnti-C-mGFP-P2A-Puro-MMP 8-P326V obtained by ligating the coding gene of claim 3 between the SgfI and MluI cleavage sites of plnti-C-mGFP-P2A-Puro, and keeping the other sequences of plnti-C-mGFP-P2A-Puro unchanged.
5. A recombinant bacterium comprising the recombinant vector according to claim 4.
6. Use of a recombinant protein matrix metalloproteinase 8 mutant MMP8-P326V as defined in claim 1 or 2 for the preparation of a product for promoting skin wound healing and for inhibiting skin healing negative events such as excessive deposition of collagen fibers and excessive inflammation caused by MMP8.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310449149.7A CN116497008B (en) | 2023-04-24 | 2023-04-24 | Matrix metalloproteinase 8 mutant MMP8-P326V and encoding gene and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310449149.7A CN116497008B (en) | 2023-04-24 | 2023-04-24 | Matrix metalloproteinase 8 mutant MMP8-P326V and encoding gene and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116497008A CN116497008A (en) | 2023-07-28 |
CN116497008B true CN116497008B (en) | 2023-12-08 |
Family
ID=87327921
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310449149.7A Active CN116497008B (en) | 2023-04-24 | 2023-04-24 | Matrix metalloproteinase 8 mutant MMP8-P326V and encoding gene and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116497008B (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101238211A (en) * | 2005-08-12 | 2008-08-06 | 普罗泰拉有限公司 | Use of matrix metalloproteinases, mutated and not mutated, for the preparation of pharmaceutical compositions, and mutated metalloproteinases with increased stability |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020155113A1 (en) * | 2001-03-13 | 2002-10-24 | Miyoung Chun | Methods for treating or preventing cardiovascular disorders by modulating metalloprotease function |
-
2023
- 2023-04-24 CN CN202310449149.7A patent/CN116497008B/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101238211A (en) * | 2005-08-12 | 2008-08-06 | 普罗泰拉有限公司 | Use of matrix metalloproteinases, mutated and not mutated, for the preparation of pharmaceutical compositions, and mutated metalloproteinases with increased stability |
Non-Patent Citations (3)
Title |
---|
Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagenesis;Vera Knäuper et al.;FEBS Letters;全文 * |
Analysis of the matrix metalloproteinase family reveals that MMP8 is often mutated in melanoma;Lavanya H Palavalli;NATURE GENETICS;全文 * |
Venter,J.C. et al..matrix metallopeptidase 8 (neutrophil collagenase) [Homo sapiens].GenBank: EAW67027.1.2015,全文. * |
Also Published As
Publication number | Publication date |
---|---|
CN116497008A (en) | 2023-07-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112877294B (en) | Preparation and application of gene-modified mesenchymal stem cell exosome | |
JP6282270B2 (en) | Neutral protease derived from recombinant Paenibacillus polymixer | |
CN116497008B (en) | Matrix metalloproteinase 8 mutant MMP8-P326V and encoding gene and application thereof | |
Gottsch et al. | Defensin gene expression in the cornea | |
CN113073081A (en) | bFGF mesenchymal stem cell exosome and preparation method and application thereof | |
CN112402592B (en) | Application of SPINK7 protein in preparation of medicine for promoting healing of excessive inflammatory wound surface | |
Nielsen et al. | Sequence comparison of the rDNA introns from six different species of Tetrahymena | |
WO2018171400A1 (en) | Bacterium for engineering acarbose, preparation method therefor, and application thereof | |
CN114934070A (en) | Mesenchymal stem cells and anti-inflammatory application thereof | |
CN113527523A (en) | Recombinant protein and construction method and application thereof | |
CN109293756B (en) | Protein for controlling spore yield and infection capacity of rice blast fungus | |
US7374911B2 (en) | Interleukin-1 related gene and protein | |
CN110946877A (en) | Stem cell biological product for treating liver cirrhosis and preparation method and application thereof | |
US8603992B2 (en) | Compositions comprising MG29 nucleic acids, polypeptides, and associated methods of use | |
JP3968618B2 (en) | Inducible promoter, inducible expression vector, and use thereof that can be used in Schizosaccharomyces pombe | |
CN115948466B (en) | Construction of Tollip knockout cell line and application of Tollip knockout cell line as picornaviridae virus vaccine production cell line | |
CN115154606B (en) | Application of Fc gamma R III inhibitor as target in preparation of medicines for treating pulmonary fibrosis | |
CN113480628B (en) | Guizhou wart mud eel polypeptide TK-CATH and coding gene and application thereof | |
CN117886922B (en) | Recombinant human fibronectin and expression system thereof | |
CN110904105B (en) | MSX promoter capable of being inhibited by pinctada martensii SMAD1/5 gene and application thereof | |
JP3694928B2 (en) | DNA fragment having promoter activity derived from basidiomycete Coriolus genus | |
KR20030060384A (en) | Expression system of human serum albumin-timp-2 fusion protein and recombinant human serum albumin- timp-2 protein | |
CN111840576A (en) | TIPE-2 and NRDP1 recombinant complex and preparation method and application thereof | |
CN113425742A (en) | Application of endometrial stem cells over-expressing CTLA-4Ig/PD-L1 in preparation of drugs for treating pulmonary fibrosis | |
EP1293563B1 (en) | Inhibition of apoptosis by the expression of antisense rna of caspase-3 |
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 |