CN107254481A - RhEGF MJ hEGF gene optimization sequences and its preparation method and application - Google Patents
RhEGF MJ hEGF gene optimization sequences and its preparation method and application Download PDFInfo
- Publication number
- CN107254481A CN107254481A CN201710451757.6A CN201710451757A CN107254481A CN 107254481 A CN107254481 A CN 107254481A CN 201710451757 A CN201710451757 A CN 201710451757A CN 107254481 A CN107254481 A CN 107254481A
- Authority
- CN
- China
- Prior art keywords
- hegf
- rhegf
- gene
- sequence
- optimization
- 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.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 59
- 238000005457 optimization Methods 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 14
- 241000894006 Bacteria Species 0.000 claims abstract description 8
- 108091008146 restriction endonucleases Proteins 0.000 claims description 14
- 239000013612 plasmid Substances 0.000 claims description 10
- 241000588724 Escherichia coli Species 0.000 claims description 9
- 238000007792 addition Methods 0.000 claims description 8
- 150000001413 amino acids Chemical class 0.000 claims description 8
- 230000006798 recombination Effects 0.000 claims description 8
- 108020004705 Codon Proteins 0.000 claims description 7
- 238000005215 recombination Methods 0.000 claims description 6
- 210000003491 skin Anatomy 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 5
- 230000029087 digestion Effects 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 238000003786 synthesis reaction Methods 0.000 claims description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 108010042407 Endonucleases Proteins 0.000 claims description 2
- 102000004533 Endonucleases Human genes 0.000 claims description 2
- 210000002615 epidermis Anatomy 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 16
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 6
- 230000004071 biological effect Effects 0.000 abstract description 5
- 230000009465 prokaryotic expression Effects 0.000 abstract description 4
- 229920001184 polypeptide Polymers 0.000 abstract description 3
- 239000002537 cosmetic Substances 0.000 abstract description 2
- 239000000969 carrier Substances 0.000 abstract 1
- 238000012215 gene cloning Methods 0.000 abstract 1
- 102000009024 Epidermal Growth Factor Human genes 0.000 description 10
- 101710098940 Pro-epidermal growth factor Proteins 0.000 description 10
- 210000003000 inclusion body Anatomy 0.000 description 9
- 230000006698 induction Effects 0.000 description 7
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 5
- 229910000162 sodium phosphate Inorganic materials 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 3
- 102400001368 Epidermal growth factor Human genes 0.000 description 3
- 101800003838 Epidermal growth factor Proteins 0.000 description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000012149 elution buffer Substances 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 239000000370 acceptor Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 210000000744 eyelid Anatomy 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000012239 gene modification Methods 0.000 description 2
- 230000005017 genetic modification Effects 0.000 description 2
- 235000013617 genetically modified food Nutrition 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 241000025995 Chone Species 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108010053070 Glutathione Disulfide Proteins 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 101500025419 Homo sapiens Epidermal growth factor Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 101000851196 Mus musculus Pro-epidermal growth factor Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000015336 Nerve Growth Factor Human genes 0.000 description 1
- 241001597008 Nomeidae Species 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 206010040943 Skin Ulcer Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000001142 circular dichroism spectrum Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 1
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000027119 gastric acid secretion Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 229940116978 human epidermal growth factor Drugs 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 1
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 description 1
- 229940053128 nerve growth factor Drugs 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000008470 skin growth Effects 0.000 description 1
- 231100000019 skin ulcer Toxicity 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000000472 traumatic effect Effects 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
-
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Animal Behavior & Ethology (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Epidemiology (AREA)
- Birds (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Crystallography & Structural Chemistry (AREA)
- Dermatology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides a kind of rhEGF MJ hEGF gene optimization sequences and its preparation method and application, Prepare restructuring hEGF is connected with hEGF particular by by MJ small peptides, obtain rhEGF MJ hEGF gene optimization sequences, and by this gene cloning to pET 30a carriers, obtain the mj hEGF expression of polypeptides engineering bacterias of high expression, Prepare restructuring hEGF, plays biological activity.RhEGF MJ hEGF gene optimization sequences expression quantity in prokaryotic expression system that the present invention is provided is significantly improved, industrialization to EGF and significant in the application of cosmetics and biomedicine field from now on.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of rhEGF MJ-hEGF gene optimizations
Sequence and its preparation method and application.
Background technology
EGF is second growth factor found after nerve growth factor, and nineteen fifty-nine is led from mouse first by Chone
Separate, because the eruption of mouse front tooth and eyelid can be promoted to open, be initially referred to as " the tooth eyelid factor " in lower gland.Then
It was found that this polypeptide can promote epidermal growth, but be named as EGF (i.e. mouse EGF,
mEGF)121.Gergoyr in 1975 extracts a kind of material with gastric acid secretion inhibiting referred to as anthelone from human urine
(urogastrone), confirmed that anthelone was people EGF (hEGF) later.
HEGF contains 53 amino acid residues, and molecular weight is 6200Da, sedimentation coefficient 1.255, isoelectric pH 4.7, containing 3
Intrachain disulfide bond, is minimum in many skin growth factors one.Relative to other many skin class materials, EGF is more steady to acid and heat
It is fixed, it is one of known most stable of polypeptide.The single-stranded many skins of hEGF systems one, circular dichroism spectra research shows that hEGF does not have α spiral shells
Rotation, β lamellar structures account for 22%.In whole hEGF molecules, two domains are mainly folded into：1-32 amino acid residues region is
N- terminal domains, 33-53 amino acid residues region is C- terminal domains.Wherein 22-32 residues area and 33-53 residues area is direct
EGF and the combination of its acceptor are participated in, the former plays Role in Plant Signal Transduction, and the latter plays stabilization.
MJ active peptides contain 21 amino acid, main nonpolar amino acid, and polar amino acid ratio is less than 20%.Non- pole
Property active region can stablize hEGF expression, and improve the stability after expression；N-terminal is added in, signal peptide can be played
Effect, hEGF is guided to cell surface rapidly, combined with EGFR acceptors.
HEGF can promote the healing of burn, wound and surgery wound, in burn, wound, skin and corneal transplantation, traumatic skin
Played an important role in the treatment such as skin ulcer.EGF is also the additive of some cosmetics, with larger market value.It is sharp at present
The major drawbacks that Urogastrone (rhEGF) is expressed with prokaryotic system are that expression quantity is relatively low, and bioactivity is not high, the eukaryotic expression cycle
Long, cumbersome, the shortcomings of cost is high is difficult large-scale production.
The content of the invention
The technical problem of solution：Present invention aim to address existing prokaryotic system expression Urogastrone (rhEGF) expression
There is provided a kind of rhEGF MJ- for the amount technical problem that relatively low, bioactivity is not high, cumbersome, cost is high
HEGF gene optimization sequences and its preparation method and application.
Technical scheme：RhEGF MJ-hEGF gene optimization sequences, as shown in SEQIDNO.1.
The preparation method of above-mentioned rhEGF MJ-hEGF gene optimization sequences, comprises the following steps：
Step 1, hEGF's hEGF genes are obtained in gene database；
Step 2, gene optimization：E. coli codon preference is carried out to the code area of hEGF's hEGF genes
Property gene optimization obtain optimization, as shown in SEQIDNO.2, optimization coding amino acid and hEGF
The consensus amino acid sequence of hEGF gene codes formation；
Step 3, genetic recombination：By optimization and MJ active peptide nucleotide sequences, as shown in SEQIDNO.3, weighed
Group, obtains rhEGF MJ-hEGF gene optimization sequences；
Step 4, restriction enzyme site is added：In 5 ' end additions of rhEGF MJ-hEGF gene optimization sequences
A kind of restriction enzyme site of restriction endonuclease, in 3 ' end another endonuclease digestion sites of addition of recombination sequence, obtains composition sequence；
Step 5, gene chemical synthesis：Full genome synthesis is carried out to obtained composition sequence.
Further, in step 3,5 ' end addition NdeI restriction enzyme sites of recombination sequence, 3 ' end additions of recombination sequence
XhoI restriction enzyme sites.
Plasmid containing above-mentioned rhEGF MJ-hEGF gene optimization sequences.
Further, the plasmid is pET-30a plasmids.
Further, above-mentioned rhEGF MJ-hEGF gene optimization sequences are inserted into pET-30a plasmids
Between Nde I restriction enzyme sites and Xho I restriction enzyme sites.
Prokaryotes containing above-mentioned rhEGF MJ-hEGF gene optimization sequences.
Further, the prokaryotes are Escherichia coli.
The hEGF hEGF that above-mentioned rhEGF MJ-hEGF gene optimization sequence tables are reached
Application in skin care item are prepared.
Beneficial effect：Protokaryon can be realized using the rhEGF MJ-hEGF gene optimizations sequence of the present invention
The high expression of bacterial host engineering bacteria EGF albumen, and with higher biological activity：1st, its expression quantity accounts for total protein
30% or so, expressed in the form of inclusion body, inclusion body processing is relative under the successful precursor of renaturation is easier to；2nd, purifying is passed through
Afterwards, EGF small peptide of the purity 95% or so is obtained, and according to the recombinant human epidermal growth factor life in Chinese Pharmacopoeia 2010 editions
Thing activity determination method, have detected biological activity, and activity is more than standards of pharmacopoeia 1 × 105UI。
Brief description of the drawings
Fig. 1 identifies Ago-Gel figure for the restructuring MJ-hEGF recombinant vectors digestion containing people in embodiment 2；
Fig. 2 is people's restructuring MJ-hEGF protein purification SDS-PAGEs in embodiment 2；
Fig. 3 is the MTT cytoactive absorbance detection results of people's restructuring MJ-hEGF albumen in embodiment 3.
Embodiment
Invention is described in further detail below by embodiment.But it will be understood to those of skill in the art that under
Row embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.
Embodiment 1
RhEGF MJ-hEGF gene preparation methods
GeneBank is logged in, the gene order of hEGF's hEGF active peptides is searched, its length nucleic acid is
159bp, hEGF are eucaryote peptide sequence, it is contemplated that follow-up research and development are expressed in protokaryon, therefore above-mentioned hEGF is true
The coding codon of core peptide sequence is compared analysis with Escherichia coli Preference codon.According to the degenerate of codon and
Do not change hEGF active peptide sequence of amino acid it is constant in the case of, with reference to biosoftware Vector NTI Suitor 7.0
HEGF nucleotide sequences are analyzed, genetic modification optimization are carried out to known hEGF, mainly by hEGF protogene sequences
The rare codon of Escherichia coli makes the codon of preference into, to improve high level expression of the target gene in Escherichia coli.And
Transgenosis checking is carried out, the optimization of hEGF genes is finally given as shown in SEQIDNO.2.
According to the degenerate of codon and do not change hEGF active peptide sequence of amino acid it is constant in the case of, with reference to raw
Thing software Vector NTI Suitor 7.0 carry out genetic modification restructuring to the optimization of hEGF genes, mainly by MJ's
Gene order (shown in SEQIDNO.3) and the optimization of hEGF's hEGF genes are recombinated, to improve target gene
Activity after the high level expression of Escherichia coli and expression.And transgenosis checking is carried out, finally give the restructuring of hEGF genes
Sequence MJ-hEGF, as shown in SEQIDNO.1.
RhEGF MJ-hEGF gene optimization sequence N-terminals and C-terminal are added into Nde I and the enzymes of Xho I respectively
Enzyme site.Serve Hai Shenggong companies and carry out full genome synthesis, and be connected into prokaryotic expression carrier pET-30a (Military Medical Science Institutes
Present) Nde I and the restriction enzyme sites of Xho I between, the prokaryotic expression plasmid pET-30a-MJ-hEGF recombinated.
Embodiment 2
The acquisition of 2.1 expression bacterial strains
Obtained prokaryotic expression carrier pET-30a-MJ-hEGF in embodiment 1 is transferred to Escherichia coli by heat shock method
In BL21 (DE3) (being purchased from Tiangeng biotech firm), kanamycins 50ug/mL is added in LB plating mediums and is screened, is chosen
After selected monoclonal digestion identification (as shown in Figure 1), carry out sequencing and further confirm that identification.
The induced expression of 2.2 rhEGF MJ-hEGF gene optimization sequences
Will recombinate positive monoclonal colony inoculation to containing concentration for 50ug/mL kanamycins LB fluid nutrient mediums in,
37 DEG C, 220rpm shaken cultivations are stayed overnight, and the bacterium solution of incubated overnight is inoculated into containing 50ug/mL cards to that is mould according to 1% ratio
In the fresh fluid nutrient medium of element, 37 DEG C, it is about 0.9 that 220rpm, which is cultivated to bacterial concentration OD600, and derivant is added immediately
IPTG to 0.5Mm, induction group and control group is continued to cultivate 3h, 12000rpm, centrifugation 10min collects thalline.
2.3 rhEGF MJ-hEGF are extracted and identification
By the thalline being collected by centrifugation, 10mL PBS gravity treatment thalline is added according to every gram of thalline weight in wet base, it is fully mixed
It is even, with final concentration of 1mg/mL 4 DEG C of digested overnight thalline of lysozyme soln.Thalline after processing surpasses in ultrasonication on ice
Sound 5s, is spaced 5s, always with 15min, then 4 DEG C, 12000rpm centrifugations 30min.Inclusion body is taken to precipitate, with 1% Triton-x-
100 washings 3 times, obtain it is thick it is pure after inclusion body be further purified.Using Ni-NTA agarose affinity chromatography post Purification of Human epidermises
Growth factor hEGF, first with 5 times of bed volume equilibrium liquid buffer solutions (8M urea, 100mM NaH2PO4, 100mM Tris-Hcl
PH=8 chromatographic column) is balanced, by flow velocity upper prop of the above-mentioned hEGF samples according to 1mL/min.After sample all complete media excessively, plus
Enter cleaning buffer solution (8M urea, 100mM NaH2PO4, 100mM Tris-Hcl pH=6.3), carry out non-specific binding impurity
Cleaning, is eventually adding elution buffer (8M urea, 100mM NaH2PO4, 100mM Tris-Hcl pH=4.5) eluted,
Elution destination protein is collected, by the destination protein after elution with the GSH containing 0.1mol/L and 0.1mol/L GSSG elution buffers
Liquid (100mM NaH2PO4, 100mM Tris-Hcl pH=8.0) under the conditions of 4 DEG C with 1：20 volume is dialysed, and 2h changes one
Secondary dialyzate, after 5 times, with elution buffer (100mM NaH2PO4, 100mM Tris-Hcl pH=8.0) and dialysis, overall process
Produced without precipitation.The SDS-PAGE of sample identifies purity of protein more than 95% after dialysis.To untreated inclusion body after induction,
The inclusion body purified after induction after thick pure inclusion body and induction carries out SDA-PAGE analyses, as a result as shown in Figure 2：M is standard
Untreated inclusion body mixture after protein molecular 1, induction, 2, after induction, the thick pure result of inclusion body washing, 3, thick after induction
Pure complete sample upper prop result after purification, is understood final to have removed out all foreign proteins on it after purification by result.
The rhEGF MJ-hEGF biological activities of embodiment 3 are verified
The Balb/c 3T3 cells (being purchased from ATCC companies) of purchase are reached into 3-4 generations, after counting, 96 orifice plates are then spread, often
Hole 1 × 105Individual cell.After (10% serum) is cultivated 24 hours in 96 orifice plate DMEM complete mediums, add and maintain culture
Continue to cultivate 24 hours in base (0.4% serum).
MJ-hEGF (final concentration of 20ng/mL), adds blank control group NC, EGF standard items (final concentration of 20ng/ respectively
ML), hEGF (final concentration of 20ng/mL) small peptide stimulates MEC Balb/c 3T3 cells after 72 hours, leads to
Cross mtt assay detection hEGF and promote cel l proliferation.ELIASA detects absorbance, absorbance and cell under 450nm wavelength
Proliferation activity is directly proportional.It is 7 × 10 to calculate specific activity7IU.As shown in following table and Fig. 3, MJ-hEGF biological activities have necessarily
Raising.
* represent that EGF treatment groups are compared with control group, with significant difference.
SEQUENCE LISTING
<110>Jiangsu Mai Jian biotechnologies Development stock Co., Ltd
<120>RhEGF MJ-hEGF gene optimization sequences and its preparation method and application
<130> 20170615
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 222
<212> DNA
<213>Artificial sequence
<400> 1
aaatacctgc tgccgaccct gctgctgctg ggtgctgccg cagcggctca gcctgcgatg 60
gcgaacagcg actctgaatg cccactgtcc cacgatggtt actgcctgca tgatggtgtg 120
tgcatgtata ttgaagctct ggacaagtat gcatgcaatt gtgttgtagg ctacatcggc 180
gagcgttgtc aataccgtga cctgaaatgg tgggaactgc gc 222
<210> 2
<211> 159
<212> DNA
<213>Artificial sequence
<400> 2
aacagcgact ctgaatgccc actgtcccac gatggttact gcctgcatga tggtgtgtgc 60
atgtatattg aagctctgga caagtatgca tgcaattgtg ttgtaggcta catcggcgag 120
cgttgtcaat accgtgacct gaaatggtgg gaactgcgc 159
<210> 3
<211> 63
<212> DNA
<213>Artificial sequence
<400> 3
aaatacctgc tgccgaccct gctgctgctg ggtgctgccg cagcggctca gcctgcgatg 60
gcg 63
Claims (9)
1. rhEGF MJ-hEGF gene optimization sequences, it is characterised in that：The sequence such as SEQIDNO.1 institutes
Show.
2. the preparation method of the rhEGF MJ-hEGF gene optimization sequences described in claim 1, its feature exists
In：Comprise the following steps：
Step 1, hEGF's hEGF genes are obtained in gene database；
Step 2, gene optimization：E. coli codon preferences are carried out to the code area of hEGF's hEGF genes
Gene optimization obtains optimization, as shown in SEQIDNO.2, the amino acid and hEGF hEGF of optimization coding
The consensus amino acid sequence of gene code formation；
Step 3, genetic recombination：By optimization and MJ active peptide nucleotide sequences, as shown in SEQIDNO.3, recombinated, obtained
To rhEGF MJ-hEGF gene optimization sequences；
Step 4, restriction enzyme site is added：It is a kind of in 5 ' end additions of rhEGF MJ-hEGF gene optimization sequences
The restriction enzyme site of restriction endonuclease, in 3 ' end another endonuclease digestion sites of addition of recombination sequence, obtains composition sequence；
Step 5, gene chemical synthesis：Full genome synthesis is carried out to obtained composition sequence.
3. the preparation method of rhEGF MJ-hEGF gene optimization sequences according to claim 2, it is special
Levy and be：In step 3,5 ' end addition NdeI restriction enzyme sites of recombination sequence, 3 ' end addition XhoI digestions positions of recombination sequence
Point.
4. the plasmid containing the rhEGF MJ-hEGF gene optimization sequences described in claim 1.
5. the rhEGF MJ-hEGF gene optimizations according to claim 4 containing described in claim 1
The plasmid of sequence, it is characterised in that：The plasmid is pET-30a plasmids.
6. the rhEGF MJ-hEGF gene optimizations according to claim 5 containing described in claim 1
The plasmid of sequence, it is characterised in that：Above-mentioned rhEGF MJ-hEGF gene optimization sequences are inserted into pET-30a
Between the Nde I restriction enzyme sites and Xho I restriction enzyme sites of plasmid.
7. the prokaryotes containing the rhEGF MJ-hEGF gene optimization sequences described in claim 1.
8. the rhEGF MJ-hEGF gene optimizations according to claim 7 containing described in claim 1
The prokaryotes of sequence, it is characterised in that：The prokaryotes are Escherichia coli.
9. people's epidermis life that the rhEGF MJ-hEGF gene optimization sequence tables described in claim 1 are reached
Applications of the long factor hEGF in skin care item are prepared.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710451757.6A CN107254481A (en) | 2017-06-15 | 2017-06-15 | RhEGF MJ hEGF gene optimization sequences and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710451757.6A CN107254481A (en) | 2017-06-15 | 2017-06-15 | RhEGF MJ hEGF gene optimization sequences and its preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107254481A true CN107254481A (en) | 2017-10-17 |
Family
ID=60023811
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710451757.6A Pending CN107254481A (en) | 2017-06-15 | 2017-06-15 | RhEGF MJ hEGF gene optimization sequences and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107254481A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022183952A1 (en) * | 2021-03-04 | 2022-09-09 | 西南大学 | Bombyx mori silk gland recombinant expression vector expressing human epidermal growth factor, preparation method therefor, and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978231A (en) * | 2012-08-02 | 2013-03-20 | 天津强微特生物科技有限公司 | Construction and screening method for recombinant human epidermal growth factor engineering bacteria |
| AU2009206089B2 (en) * | 2008-01-15 | 2014-08-28 | Abbvie Inc. | Improved mammalian expression vectors and uses thereof |
| CN105463006A (en) * | 2014-09-12 | 2016-04-06 | 尹成凯 | Novel preparation method for recombinant human epidermal growth factor |
-
2017
- 2017-06-15 CN CN201710451757.6A patent/CN107254481A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2009206089B2 (en) * | 2008-01-15 | 2014-08-28 | Abbvie Inc. | Improved mammalian expression vectors and uses thereof |
| CN102978231A (en) * | 2012-08-02 | 2013-03-20 | 天津强微特生物科技有限公司 | Construction and screening method for recombinant human epidermal growth factor engineering bacteria |
| CN105463006A (en) * | 2014-09-12 | 2016-04-06 | 尹成凯 | Novel preparation method for recombinant human epidermal growth factor |
Non-Patent Citations (4)
| Title |
|---|
| DAVID RHYS THOMAS等: "Improved expression of recombinant plant-made hEGF", 《PLANT CELL REP》 * |
| 史济平主编: "《药学分子生物学》", 31 July 2007 * |
| 吕正兵等: "家蚕表达人表皮生长因子gp67信号肽融合蛋白及生物活性的研究", 《生物工程学报》 * |
| 张海淼等: "人表皮生长因子融合蛋白的表达及纯化工艺的优化", 《中国生物工程杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022183952A1 (en) * | 2021-03-04 | 2022-09-09 | 西南大学 | Bombyx mori silk gland recombinant expression vector expressing human epidermal growth factor, preparation method therefor, and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110845603A (en) | Human collagen 17-type polypeptide, production method and use thereof | |
| WO2017069158A1 (en) | Immunoglobulin-binding polypeptide | |
| CN114853881A (en) | Recombinant human fusion collagen and efficient hydroxylation method and application thereof | |
| CN113087804A (en) | Bivalent plant immune fusion protein and production method and application thereof | |
| CN118047857A (en) | Preparation method of biological synthetic human body structural material | |
| CN116333097A (en) | High-activity recombinant human fibronectin and preparation method and application thereof | |
| CN103710367B (en) | A kind of recombined human kallikrein 1 and encoding gene thereof and preparation method | |
| CN107254472A (en) | HEGF's hEGF gene optimization sequences and its preparation method and application | |
| CN102666570A (en) | Novel peptidyl alpha-hydroxyglycine alpha-amidating lyases | |
| CN107254481A (en) | RhEGF MJ hEGF gene optimization sequences and its preparation method and application | |
| CN108368512B (en) | Preparation method of recombinant human granulocyte colony stimulating factor | |
| CN101605807A (en) | The expression system for recombinant human arginase i of improvement | |
| CN116554309A (en) | Recombinant human III type collagen and preparation method and application thereof | |
| CN102021173A (en) | Preparation method for soluble truncated human tumor necrosis factor-related apoptosis inducing ligand (TRAIL) active protein | |
| CN107435045B (en) | A kind of nucleotide sequence and solution expression with high efficiency method of optimum combination Human Inter Leukin-2 | |
| CN101433713B (en) | GnRH-PE mutant fusion protein and uses thereof | |
| CN103360497A (en) | Novel antitumor fusion protein vaccine, and preparation method and application thereof | |
| CN110079539B (en) | Preparation method of prostatic acid phosphatase/granulocyte-macrophage colony stimulating factor | |
| CN110004153A (en) | Chicken recombinant C EBP γ albumen, its DNA sequences encoding and application | |
| CN116478969A (en) | Collagenase mutant, method for promoting secretory expression of recombinant collagenase and application thereof | |
| CN101608179B (en) | Fusion heparinase and coding gene thereof | |
| CN114908113A (en) | Preparation method of human interleukin-5 recombinant protein | |
| CN112646044B (en) | TFF2-Fc fusion protein and high-efficiency expression production method thereof | |
| CN108484749A (en) | A kind of recombinant soluble human source Bone targeting gamma interferon 1-b and preparation method thereof | |
| CN110305887A (en) | Anti-fungus peptide Drosomycin, preparation method and applications |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171017 |
|
| RJ01 | Rejection of invention patent application after publication |