WO2020253287A1 - 优化的fgf2基因功能区序列及其在制备人fgf2中的应用 - Google Patents
优化的fgf2基因功能区序列及其在制备人fgf2中的应用 Download PDFInfo
- Publication number
- WO2020253287A1 WO2020253287A1 PCT/CN2020/079897 CN2020079897W WO2020253287A1 WO 2020253287 A1 WO2020253287 A1 WO 2020253287A1 CN 2020079897 W CN2020079897 W CN 2020079897W WO 2020253287 A1 WO2020253287 A1 WO 2020253287A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- fgf2
- construct
- fgf
- dna sequence
- seq
- Prior art date
Links
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 101150019331 FGF2 gene Proteins 0.000 title description 21
- 241000282414 Homo sapiens Species 0.000 title description 9
- YSFTYXKQUONNFY-NQXPEFQPSA-N fgf2 Chemical compound C([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](C)NC(=O)CNC(=O)[C@H](CO)NC(=O)CNC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H]1NCCC1)C1=CC=CC=C1 YSFTYXKQUONNFY-NQXPEFQPSA-N 0.000 title description 9
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims abstract description 128
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims abstract description 128
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 44
- 239000013600 plasmid vector Substances 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 29
- 230000000295 complement effect Effects 0.000 claims abstract description 25
- 210000004027 cell Anatomy 0.000 claims description 48
- 238000000746 purification Methods 0.000 claims description 27
- 230000017730 intein-mediated protein splicing Effects 0.000 claims description 23
- 108091008146 restriction endonucleases Proteins 0.000 claims description 23
- 239000012634 fragment Substances 0.000 claims description 20
- 210000004962 mammalian cell Anatomy 0.000 claims description 17
- 239000013612 plasmid Substances 0.000 claims description 17
- 238000001042 affinity chromatography Methods 0.000 claims description 11
- 238000001890 transfection Methods 0.000 claims description 7
- 210000004748 cultured cell Anatomy 0.000 claims description 5
- 102000003960 Ligases Human genes 0.000 claims description 4
- 108090000364 Ligases Proteins 0.000 claims description 4
- 230000001404 mediated effect Effects 0.000 claims description 4
- 239000000284 extract Substances 0.000 claims description 3
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 2
- 229960002897 heparin Drugs 0.000 claims description 2
- 229920000669 heparin Polymers 0.000 claims description 2
- 108010013829 alpha subunit DNA polymerase III Proteins 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 19
- 230000003247 decreasing effect Effects 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 description 47
- 102000004169 proteins and genes Human genes 0.000 description 38
- 238000006243 chemical reaction Methods 0.000 description 20
- 239000000047 product Substances 0.000 description 19
- 238000004458 analytical method Methods 0.000 description 17
- 239000002609 medium Substances 0.000 description 14
- 108020004414 DNA Proteins 0.000 description 11
- 239000013598 vector Substances 0.000 description 11
- 102000007528 DNA Polymerase III Human genes 0.000 description 10
- 108010071146 DNA Polymerase III Proteins 0.000 description 10
- 230000004071 biological effect Effects 0.000 description 10
- 239000000872 buffer Substances 0.000 description 10
- 230000029087 digestion Effects 0.000 description 10
- 230000002441 reversible effect Effects 0.000 description 9
- 238000012408 PCR amplification Methods 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 229960000723 ampicillin Drugs 0.000 description 8
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 238000001976 enzyme digestion Methods 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 7
- 239000012154 double-distilled water Substances 0.000 description 7
- 239000000499 gel Substances 0.000 description 7
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 7
- 239000002158 endotoxin Substances 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 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
- 102000012410 DNA Ligases Human genes 0.000 description 5
- 108010061982 DNA Ligases Proteins 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000003776 cleavage reaction Methods 0.000 description 5
- 239000003623 enhancer Substances 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 230000007017 scission Effects 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 102000005636 Cyclic AMP Response Element-Binding Protein Human genes 0.000 description 4
- 108010045171 Cyclic AMP Response Element-Binding Protein Proteins 0.000 description 4
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 4
- 101100281008 Homo sapiens FGF2 gene Proteins 0.000 description 4
- 238000000134 MTT assay Methods 0.000 description 4
- 231100000002 MTT assay Toxicity 0.000 description 4
- 108010057466 NF-kappa B Proteins 0.000 description 4
- 102000003945 NF-kappa B Human genes 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- 239000011543 agarose gel Substances 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000000508 neurotrophic effect Effects 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000376 reactant Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical group CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 239000013592 cell lysate Substances 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000014511 neuron projection development Effects 0.000 description 3
- 230000004481 post-translational protein modification Effects 0.000 description 3
- 230000002035 prolonged effect Effects 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 238000007480 sanger sequencing Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 208000002109 Argyria Diseases 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 102000005720 Glutathione transferase Human genes 0.000 description 2
- 108010070675 Glutathione transferase Proteins 0.000 description 2
- 101001052030 Mus musculus Fibroblast growth factor 2 Proteins 0.000 description 2
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 2
- 230000033115 angiogenesis Effects 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 238000004166 bioassay Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 210000003000 inclusion body Anatomy 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000012723 sample buffer Substances 0.000 description 2
- 229910052709 silver Inorganic materials 0.000 description 2
- 239000004332 silver Substances 0.000 description 2
- 238000001542 size-exclusion chromatography Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000012130 whole-cell lysate Substances 0.000 description 2
- UHEPSJJJMTWUCP-DHDYTCSHSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2s,3r,4r,5s,6r)-3-amino-4,5-dihydroxy-6-[(1r)-1-hydroxyethyl]oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol;sulfuric acid Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H]([C@@H](C)O)O2)N)[C@@H](N)C[C@H]1N UHEPSJJJMTWUCP-DHDYTCSHSA-N 0.000 description 1
- AXAVXPMQTGXXJZ-UHFFFAOYSA-N 2-aminoacetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound NCC(O)=O.OCC(N)(CO)CO AXAVXPMQTGXXJZ-UHFFFAOYSA-N 0.000 description 1
- 108091006112 ATPases Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 102000057290 Adenosine Triphosphatases Human genes 0.000 description 1
- 102000000584 Calmodulin Human genes 0.000 description 1
- 108010041952 Calmodulin Proteins 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101001052035 Homo sapiens Fibroblast growth factor 2 Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 101710128836 Large T antigen Proteins 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000894763 Nostoc punctiforme PCC 73102 Species 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
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- QTENRWWVYAAPBI-YZTFXSNBSA-N Streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.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](N=C(N)N)[C@@H](O)[C@H](N=C(N)N)[C@@H](O)[C@@H]1O.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](N=C(N)N)[C@@H](O)[C@H](N=C(N)N)[C@@H](O)[C@@H]1O QTENRWWVYAAPBI-YZTFXSNBSA-N 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- HPNSNYBUADCFDR-UHFFFAOYSA-N chromafenozide Chemical compound CC1=CC(C)=CC(C(=O)N(NC(=O)C=2C(=C3CCCOC3=CC=2)C)C(C)(C)C)=C1 HPNSNYBUADCFDR-UHFFFAOYSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000037011 constitutive activity Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 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 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000011143 downstream manufacturing Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000009931 harmful effect Effects 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000005040 ion trap Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002297 mitogenic effect Effects 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000004766 neurogenesis Effects 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 229920002704 polyhistidine Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 238000000734 protein sequencing Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000036303 septic shock Effects 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
Definitions
- the present invention relates to a DNA sequence for expressing FGF-2, a construct containing the DNA sequence, a method for preparing the construct, the use of the construct in expressing FGF-2, and a method for preparing FGF- 2 method.
- FGF2 is a very valuable protein in the pharmaceutical and healthcare industries.
- FGF2 is an effective therapeutic protein for treating neurodegenerative diseases, heart disease, angiogenesis, difficult-to-heal wounds and fractures; it also plays an important role in the mass production of stem cells.
- E. coli Due to the low preparation cost, high replication rate, and high productivity, E. coli has been used for the purification of recombinant proteins for a long time [1-4].
- the use of prokaryotic expression systems often encounter obstacles in the purification of mammalian proteins [5,6].
- scientists have been using eukaryotic hosts such as yeast and insect cells to overcome these limitations [7]. Nonetheless, it seems intuitive to use cultured cells of human origin for protein expression of human origin. In fact, there is an upward trend in the use of mammalian cells to produce recombinant proteins [8,9].
- affinity tags such as polyhistidine (His-tag), glutathione S-transferase (GST) or calmodulin-binding peptide (CBP) to the target protein is a common strategy for protein purification [10].
- the label provides a simple and simplified purification scheme for crude cell lysates. Although the tag can be removed after purification, it usually leaves some extra amino acids on the cleaved protein derivative. Although these extra amino acids are few, they can change the folding and thus change its natural form [11-14]. Recombinant proteins with extra and unwanted amino acids may cause irreversible harmful effects, bind off-target receptors, and may cause cancer [15,16] and allergies [16,17].
- Inteins are self-cleaving elements in naturally occurring proteins.
- VMA gene encodes vascular ATPase, in which the intein is cleaved, and then the N-terminal and C-terminal exons are connected to produce a functional full-length protein [19-24].
- inteins have been found in many organisms, scientists have used this self-excision property in the field of protein purification. When properly designed, the site-specific and precise cleavage of the intein can produce the same protein product as humans [23,25].
- an aspect of the present invention provides a DNA sequence for expression of FGF-2, wherein the sequence shown in SEQ ID NO.1: CCGGCTCTCCCAGAGGATGGCGGCTCAGGAGCCTTTCCACCAGGACACTTCAAAGATCCGAAGAGGCTTTACTGCAAGAATGGTGGATTTTTCCTCCGCATCCATCCAGACGGTCGGGTGGACGGCGTACGGGAGAAATCCGATCCGCATATAAAGCTGCAGCTGCAAGCTGAAGAACGAGGGGTGGTTAGCATAAAGGGCGTGTGTGTGCTAATAGGTACCTTGCCATGAAAGAAGACGGACGGCTCCTCGCTTCTAAGTGCGTGACCGACGAGTGCTTCTTCTTTGAGCGGCTAGAGTCAAACAATTATAACACCTATAGGTCAAGAAAGTATACGAGCTGGTACGTTAAGCGGACCGGCCAGTACAAGCTTGGTAGCAAAACAGGCCCTGGCCAGAAGGCTATCCTCTTCCTATGAGTGCC
- the present invention also provides another DNA sequence for expressing FGF-2, which is characterized in that the sequence is shown in SEQ ID NO. 2:
- the present invention provides a construct characterized in that the construct includes the DNA sequence shown in SEQ ID NO. 1 between the restriction enzyme cut sites of the plasmid vector EcoRI and NotI or its complementary sequence , Or the construct includes the DNA sequence shown in SEQ ID NO. 2 between the plasmid vector EcoRI and NotI restriction site or its complementary sequence.
- the present invention provides a method for preparing a construct, which is characterized in that the method includes the following steps:
- ligase Use ligase to connect the DNA sequence obtained in step a or its complementary sequence with the large fragment obtained in step b.
- step d Transfect cells with the ligation product obtained in step c, select positive clones, and extract plasmids.
- the present invention provides the application of the construct provided by the present invention in expressing FGF-2.
- the present invention provides a method for preparing FGF-2, which is characterized in that the method includes:
- Cultured cells cultured mammalian cells
- the DNA sequence and its construct for expressing FGF-2 provided by the present invention can significantly increase the expression level of FGF-2, and the expressed FGF-2 is basically soluble, and its biological activity is comparable to that of commercially available FGF-2. The biological activity is comparable. Therefore, using the construct provided by the present invention to express FGF-2 can significantly increase the expression of FGF-2 with biological activity, thereby greatly reducing the cost of preparing and purifying FGF-2.
- Figure 1 is a schematic diagram of the FGF2 construct and the his-DnaE-FGF2 construct.
- A Synthesize the DNA of the mature functional fragment of the fgf2 gene and clone it into the pcDNA 3.1 (+) vector under the control of the CMV enhancer/promoter to form a pcDNA3.1-FGF2 construct.
- B1 Synthesize the NF- ⁇ B binding site and CREB binding site and clone them into the CMV promoter to form the pNC1 vector;
- B2 Then clone the DNA of the mature functional fragment of the fgf2 gene into the pNC1 vector to form pNC1-FGF2 Construct.
- FIG. 2 shows the expression of mature functional fragment FGF2 protein in HEK293T cells.
- A The pcDNA3.1-FGF2 and pNC1-FGF2 constructs were transfected into HEK293T cells, and the whole cell lysate was analyzed by Western blot at designated time points.
- C Analysis of supernatant (S) and pellet (P) from cell lysates by Western blot. Commercially purchased FGF2 (+ve) was used as a positive control. For the pcDNA3.1-FGF2 and pNC1-FGF2 constructs, the FGF2 expressed by HEK293T is quite soluble.
- Figure 3 is the purification of FGF2 expressed in HEK293T cells.
- A The whole cell lysate and the eluate from the size exclusion chromatography sample were subjected to SDS-PAGE and stained with Coomassie brilliant blue.
- B The sample from (A) was lyophilized and reconstituted in 0.1x PBS, and then analyzed by silver staining after SDS-PAGE. Only 1 weak band (indicated by the arrow) was observed in the purified FGF2.
- Figure 4 is a bioassay of purified FGF2.
- B Commercially purchased and purified FGF2 at a concentration of 2ng/mL was added to the medium for 3 days. Phase contrast micrographs were taken on day 0 and day 3. Prolonged neurite outgrowth (indicated by arrows) in PC12 cells was observed after 3 days of FGF2 treatment.
- Figure 5 shows the expression of mature functional fragment FGF2 protein in HEK293T cells and the purification of FGF2 by intein-assisted cleavage.
- the pNC1-6xhis-DnaE-FGF2 construct was transfected into HEK293T cells, the medium was collected and spun at 2,000 g for 10 minutes to remove cell debris, and filtered through a 0.45 ⁇ m filter.
- the protein was purified by Ni-NTA affinity chromatography. Induces the cleavage of the DnaE intein in the cleavage buffer. Starting from 2 hours after induction, the full-length his-DnaE-FGF2 protein (band indicated by *) was cleaved into FGF2 (band indicated by #). The lysis is complete 6 hours after induction.
- Figure 6 is a bioassay of purified and endoprotein-cleaved FGF2.
- B 2ng/mL purified and intein-cut FGF2 was added to the culture medium for 3 days. Phase contrast micrographs were taken on day 0 and day 3. Prolonged neurite outgrowth (indicated by arrows) in PC12 cells was observed after 3 days of FGF2 treatment.
- the present invention provides a DNA sequence for expressing FGF-2, which is characterized in that the sequence is shown in SEQ ID NO. 1:
- the present invention also provides a DNA sequence for expressing FGF-2, which is characterized in that the sequence is shown in SEQ ID NO. 2:
- the present invention also provides a construct, which is characterized in that the construct includes the DNA sequence shown in SEQ ID NO.1 between the plasmid vector EcoRI and NotI restriction site or its complementary sequence, or the The construct includes the DNA sequence shown in SEQ ID NO. 2 between the plasmid vector EcoRI and the NotI restriction site or its complementary sequence.
- the plasmid vector is pcDNA 3.1(+) or pNC1, more preferably pNC1.
- the construct includes the DNA shown in SEQ ID NO. 2 between the plasmid vector EcoRI and NotI restriction enzyme cutting sites The sequence or its complementary sequence, and the plasmid vector is pNC1.
- the present invention also provides a method for preparing the construct, which is characterized in that the method includes the following steps:
- ligase Use ligase to connect the DNA sequence obtained in step a or its complementary sequence with the large fragment obtained in step b.
- step d Transfect cells with the ligation product obtained in step c, select positive clones, and extract plasmids.
- the plasmid vector is pcDNA 3.1(+) or pNC1, more preferably pNC1.
- the present invention also provides the use of the construct in expressing FGF-2.
- the present invention also provides a method for preparing FGF-2, which is characterized in that the method includes:
- Cultured cells cultured mammalian cells
- the affinity chromatography is performed through a heparin binding domain.
- step b the mammalian cells obtained in step a are transfected with the above-mentioned construct; the affinity chromatography is an affinity mediated by intein DnaE And chromatography.
- mammalian cells are not particularly limited, for example, HEK293T, HeLa, C2C12, etc. can be used.
- the mammalian cell is HEK 293T.
- PCR using GeneArt Strings gene synthesis service (Thermo-Fisher Scientific, Waltham, MA) to synthesize the enhancer sequence, which is the DNA sequence containing the NF- ⁇ B binding site and the DNA sequence of the CREB binding site DNA sequence;
- pcDNA 3.1(+) was purchased from Thermo Scientific, USA, and its vector map is shown in Figure 1A. Restriction endonucleases NheI and HindIII, and T4 DNA Ligase were purchased from NEB. Plasmid extraction kit and DNA fragment recovery kit were purchased from Thermo Scientific.
- the enhancer template is synthesized by GeneArt string service (thermos scientific), and its DNA sequence is shown in SEQ ID NO. 5: 5'GGAAATCCCCGGAAATCCCCGTAAAATTTGCGTCAACACTGCTCAAC.
- the forward and reverse primers used are shown in Table 1. Add the following reactants in the EP tube in sequence: 0.5 ⁇ l template, 1x buffer, 1 ⁇ M forward primer, 1 ⁇ M reverse primer, 0.5 ⁇ l polymerase, 4 ⁇ l dNTP, add ddH2O to 50 ⁇ l reaction system for reaction.
- Reaction conditions 95°C, 5min; 95°C, 15s; 55°C, 10s; 72°C, 10s; 35 cycles; 72°C extension for 10min, 4°C to terminate the reaction.
- the PCR amplification product was electrophoresed in a 3% agarose gel for result detection, and the inserted band was recovered with a DNA gel recovery and purification kit. A 59bp fragment was obtained, which was consistent with the expected size.
- the fragments recovered by PCR and the plasmid vector pcDNA 3.1(+) were digested with NheI and HindIII, then ligated with T4DNA ligase, and the ligated product was transformed into chemically competent cell DH5a strain, and then coated on ampicillin resistant solid medium On a petri dish, pick several monoclonal colonies and inoculate them into the ampicillin-resistant liquid medium, and cultivate overnight on a constant temperature shaker at 37°C and 250 rpm with shaking.
- the plasmid was extracted with a plasmid extraction kit to obtain the recombinant plasmid vector pNC1.
- Recombinant plasmid DNA 5 ⁇ L, 10 ⁇ buffer 2 ⁇ L, NheI 0.5 ⁇ L, HindIII 0.5 ⁇ L, ddH2O 11 ⁇ L
- the total volume is 20 ⁇ L. 37°C, 15min.
- the positive clones verified by restriction digestion were selected for sequencing analysis, and the results were analyzed by FinchTV and compared with BLAST software to verify the correctness of the recombinant plasmid vector.
- the analysis results showed that the enhancer nucleotide sequence was completely correct, and the recombinant plasmid vector pNC1 was successfully obtained.
- FGF2-pcDNA3.1(+) construct FGF2-pNC1 construct, and his-DnaE-FGF2-pNC1 construct
- the DNA sequence (amino acids 143-288, PRO_0000008933) of the fgf2 gene without the propeptide sequence (that is, the mature functional fragment of the fgf2 gene) was designed for human codon optimization.
- the present invention uses 6x his tags and uses the DNA polymerase III (DnaE) intein of Nostoc punctiforme PCC73102 (Npu) fused with the fgf2 gene to promote human fibroblast growth factor 2 ( FGF2) purification.
- DnaE DNA polymerase III
- Npu Nostoc punctiforme PCC73102
- GeneArt Strings gene synthesis service was used to synthesize enhancer sequences containing NF- ⁇ B binding sites and CREB binding sites, and cloned into pcDNA 3.1(+) with NheI and HindIII sites (Thermo- Fisher Scientific, Waltham, MA)) to form the expression plasmid vector pNC1.
- the synthetic 6xhis-DnaE-FGF2) was cloned into pNC1 with EcoRI and NotI sites to form a his-DnaE-FGF2-pNC1 construct, as shown in Figure 1C. All sequences were confirmed by Sanger sequencing.
- Antibodies used in Western blotting mouse FGF-2 (clone C-2, Santa Cruz Biotechnology, Dallas, TX), mouse anti- ⁇ -actin (Sigma, St. Louis, MO).
- FGF2-pcDNA3.1(+) construct The specific process of constructing FGF2-pcDNA3.1(+) construct is as follows:
- the template of fgf2 is synthesized by GeneArt string service (thermos scientific), and its DNA sequence is shown in SEQ ID NO.1
- the forward and reverse primers used are shown in Table 1. Add the following reactants to the EP tube in sequence: 0.5 ⁇ l template, 1x buffer, 1 ⁇ M forward primer, 1 ⁇ M reverse primer, 0.5 ⁇ l polymerase, 4 ⁇ l dNTP, add ddH2O to 50 ⁇ l reaction system for reaction.
- Reaction conditions 95°C, 5min; 95°C, 15s; 55°C, 10s; 72°C, 30min; 35 cycles; 72°C extension for 10min, 4°C to terminate the reaction.
- the PCR amplification product was electrophoresed in a 1% agarose gel for result detection, and the inserted band was recovered with a DNA gel recovery and purification kit. A 460bp fragment was obtained, which was consistent with the expected size.
- the fragments recovered by PCR and the plasmid vector pcDNA 3.1(+) were digested with EcoRI and NotI, and then ligated with T4DNA ligase.
- the ligated product was transformed into chemically competent cell DH5a strain, and then coated on ampicillin resistant solid medium On a petri dish, pick several monoclonal colonies and inoculate them into the ampicillin-resistant liquid medium, and cultivate overnight on a constant temperature shaker at 37°C and 250 rpm with shaking.
- the plasmid was extracted with a plasmid extraction kit to obtain FGF2-pcDNA3.1(+) construct.
- restriction enzyme digestion with EcoRI and NotI was used for identification.
- the restriction digestion identification system and reaction conditions are as follows:
- FGF2-pcDNA3.1(+) construct 5 ⁇ L, 10 ⁇ buffer 2 ⁇ L, EcoRI 0.5 ⁇ L, NotI 0.5 ⁇ L, ddH2O 11 ⁇ L, total volume 20 ⁇ L. 37°C, 15min.
- the positive clones verified by restriction digestion were selected for sequencing analysis, and the results were analyzed by FinchTV and compared with BLAST software to verify the correctness of the FGF2-pcDNA3.1(+) construct.
- the analysis results showed that the nucleotide sequence of fgf2 was completely correct, and the FGF2-pcDNA3.1(+) construct was successfully obtained.
- FGF2-pNC1 construct The specific process of constructing FGF2-pNC1 construct is as follows:
- the template of fgf2 is synthesized by GeneArt string service (thermos scientific), and its DNA sequence is shown in SEQ ID NO.1:
- the forward and reverse primers used are shown in Table 1. Add the following reactants in the EP tube in sequence: 0.5 ⁇ l template, 1x buffer, 1 ⁇ M forward primer, 1 ⁇ M reverse primer, 0.5 ⁇ l polymerase, 4 ⁇ l dNTP, add ddH2O to 50 ⁇ l reaction system for reaction.
- Reaction conditions 95°C, 5min; 95°C, 15s; 55°C, 10s; 72°C, 30min; 35 cycles; 72°C extension for 10min, 4°C to terminate the reaction.
- the PCR amplification product was electrophoresed in a 1% agarose gel for result detection, and the inserted band was recovered with a DNA gel recovery and purification kit. A 460bp fragment was obtained, which was consistent with the expected size.
- the fragments recovered by PCR and the plasmid vector pNC1 were digested with EcoRI and NotI, and then ligated with T4DNA ligase.
- the ligated product was transformed into chemically competent cell DH5a strain, and spread on the ampicillin resistant solid medium plate, and picked Several monoclonal colonies were inoculated into the ampicillin resistant liquid medium and cultured overnight on a constant temperature shaker at 37°C and 250 rpm with shaking.
- the plasmid was extracted with a plasmid extraction kit to obtain the pNC1 construct.
- restriction enzyme digestion with EcoRI and NotI was used for identification.
- the restriction digestion identification system and reaction conditions are as follows:
- FGF2-pNC1 construct 5 ⁇ L, 10 ⁇ buffer 2 ⁇ L, EcoRI 0.5 ⁇ L, NotI 0.5 ⁇ L, ddH 2 O 11 ⁇ L, total volume 20 ⁇ L. 37°C, 15min. After identification by restriction enzyme digestion, the product obtained is consistent with the expected analysis size.
- the positive clones verified by restriction digestion were selected for sequencing analysis, and the results were analyzed by FinchTV and compared with BLAST software to verify the correctness of the FGF2-pNC1 construct.
- the analysis results showed that the nucleotide sequence of fgf2 was completely correct, and the FGF2-pNC1 construct was successfully obtained.
- his-DnaE-FGF2-pNC1 construct is as follows:
- the template of his-DnaE-FGF2 is synthesized by GeneArt string service (thermos scientific), and its DNA sequence is shown in SEQ ID NO. 2:
- Reaction conditions 95°C, 5min; 95°C, 15s; 55°C, 10s; 72°C, 30min; 35 cycles; 72°C extension for 10min, 4°C to terminate the reaction.
- the PCR amplification product was electrophoresed in a 1% agarose gel for result detection, and the GFP band was recovered with a DNA gel recovery and purification kit. A fragment of 1097bp was obtained, which was consistent with the expected size.
- the fragments recovered by PCR and the plasmid vector his-DnaE-FGF2 were digested with EcoRI and NotI, and then ligated with T4DNA ligase.
- the ligated product was transformed into chemically competent cell DH5a strain, and then coated on ampicillin resistant solid medium On a petri dish, pick several monoclonal colonies and inoculate them into the ampicillin-resistant liquid medium, and cultivate overnight on a constant temperature shaker at 37°C and 250 rpm with shaking.
- the plasmid was extracted with a plasmid extraction kit to obtain his-DnaE-FGF2 construct.
- restriction enzyme digestion with EcoRI and NotI was used for identification.
- the restriction digestion identification system and reaction conditions are as follows:
- His-DnaE-FGF2 construct 5 ⁇ L, 10 ⁇ buffer 2 ⁇ L, EcoRI 0.5 ⁇ L, NotI 0.5 ⁇ L, ddH2O 11 ⁇ L, total volume 20 ⁇ L. 37°C, 15min.
- the positive clones verified by restriction digestion were selected for sequencing analysis, and the results were analyzed by FinchTV and compared with BLAST software to verify the correctness of the his-DnaE-FGF2 construct.
- the analysis results showed that the nucleotide sequence of his-DnaE-fgf2 was completely correct, and the his-DnaE-FGF2 construct was successfully obtained.
- the DNA containing the NheI and HindIII restriction enzyme cut site enhancers synthesized by PCR is SEQ ID NO.11 (that is, the DNA containing the NheI and HindIII restriction enzyme cut site SEQ ID NO.5):
- the DNA sequence of FGF2 containing EcoRI and NotI restriction enzyme sites synthesized by PCR is SEQ ID NO.12 (that is, SEQ ID NO.1 containing EcoRI and NotI restriction enzyme sites):
- SEQ ID NO.13 The DNA sequence of his-DnaE-FGF2 with EcoRI and NotI restriction enzyme sites synthesized by PCR is SEQ ID NO.13 (that is, SEQ ID NO. 2 containing EcoRI and NotI restriction enzyme sites):
- the HEK 293T and C2C12 cells were maintained in DMEM containing 10 vol% FBS and 1 vol% penicillin-streptomycin solution (Thermo-Fisher Scientific, Waltham, MA) at a temperature of 37° C. and containing 5% CO2.
- the PC12 cells were maintained in DMEM containing 10 vol% HS, 5% FBS and 1 vol% penicillin streptomycin solution, the temperature in the environment was 37° C. and 5% CO2.
- the pcDNA3.1-FGF2, pNC1-FGF2 and pNC1-6xhis-DnaE-FGF2 constructs were transfected into HEK 293T with Lipofectamine 2000 (Thermo-Fisher Scientific, Waltham, MA).
- the medium was collected and rotated at 2,000 g for 10 minutes to remove cell debris, and filtered through a 0.45 ⁇ m filter.
- the filtrate was then passed through a pre-packed heparin-agarose column (BioRad Laboratories, Hercules, CA) equilibrated with 50 mM Tris-HCl (pH 7.5). Wash the column thoroughly with 50mM Tris-HCl (pH 7.5), 0.2M NaCl.
- FGF2 was eluted with a NaCl gradient from 0.3M to 3M (4-5 bed volumes used for the gradient).
- the protein was passed through a pre-packed Sephadex G25 column equilibrated with 50 mM Tris-HCl (pH 7.5) and eluted with the same buffer.
- FGF2 expressed in cells the cells were washed three times with ice-cold TBS, and then sonicated in lysis buffer (TBS provided with cOmpleteTM Protease Inhibitor Cocktail). Then FGF2 is purified in the culture medium.
- the histidine-tagged DnaE-FGF2 was purified through a prepackaged Ni-NTA column. After washing, the column was incubated in 50 mM Tris-HCl (pH 6.2), 10 mM EDTA, 200 mM NaCl at 22°C for different durations of 0, 1, 2, 4, 6, and 10 hours, respectively, to induce DnaE The C-terminus of the intein is excised.
- the protein was separated in 15% by volume Tris-glycine SDS-PAGE. The gel was stained with silver to obtain purified protein. On the gel, the band corresponding to FGF2 was cut, washed and incubated with 1 ⁇ g trypsin in 50 mM NH 4 HCO 3 at 4° C. overnight. The hydrolyzed samples were analyzed by the LTQ Velos Linear Ion Trap Mass Spectrometer (Thermo Fisher Scienctific, San Jose) combined with the Accela HPLE system. Obtain a complete MS scan (300-2000m/z) and use the Mascot search engine (Matric Science, Boston, MA) to identify the peptide sequence.
- the sample was spotted or transferred to 0.2 ⁇ m NC membrane (BioRad Laboratories, Hercules, CA), and then blotted with antibody (mouse FGF-2 (clone C-2, Santa Cruz Biotechnology, Dallas, TX), Mouse anti- ⁇ -actin (Sigma, St. Louis, MO)).
- NC membrane BioRad Laboratories, Hercules, CA
- antibody mouse FGF-2 (clone C-2, Santa Cruz Biotechnology, Dallas, TX), Mouse anti- ⁇ -actin (Sigma, St. Louis, MO)
- MTT assay was performed as described above.
- C2C12 cells were seeded on a 96-well plate, and DMEM+0.5% FBS and 1ng/mL of commercial FGF2, purified FGF2 and endopeptide excised FGF2 were provided.
- the cell activity was measured by adding MTT to a final concentration of 1 mg/mL, and incubated at 37°C for 6 hours. The medium was then replaced with DMSO, and the absorbance was measured at 540 nm in a microplate reader.
- PC12 cells were cultured with a medium provided with 2ng/mL FGF2 for 3 days. Observe the morphology of the cells and take images through a phase contrast optical microscope.
- the full-length FGF2 protein consists of 288 amino acids, of which amino acids 1-142 are cleaved and removed to produce functional FGF2. Therefore, the protein sequence of amino acids 143-288 is codon optimized for use in Homo sapiens.
- the NF- ⁇ B binding site and CREB binding site enhancer sequences were cloned into the pcDNA3.1 vector containing the natural CMV immediate early promoter/enhancer sequence.
- the synthesized fgf2 gene was then cloned into the pcDNA3.1 and pNC1 vectors ( Figure 1) to compare the expression of the two clones in mammalian cells.
- pNC1 vector greatly increases the expression level of FGF2 in mammalian cells, as shown in Figure 2.
- the neomycin resistance gene also provides a selection marker through G418 sulfate. Therefore, stably transfected cells can be used to purify the same exogenous human FGF2.
- HEK293T cells were chosen for purification because of their ease of transfection.
- the SV40 large T antigen also provides cells with the ability to replicate the transfected plasmid, which contains the SV40 origin of replication.
- the CMV promoter is one of the strongest promoters and has constitutive activity in HEK293T cells.
- HEK293T cells are adherent cells, they are universally applicable to suspension culture, so they are beneficial to expand the production of FGF2.
- the pcDNA3.1-FGF2, pNC1-FGF2 and pNC1-6xhis-DnaE-FGF2 constructs were transiently transfected into HEK293T cells and the expression was monitored. Experiments show that the pNC1 vector greatly increases the expression level of FGF2 ( Figure 2A, 2B). The expression of FGF2 reached a peak 48 hours after transfection and remained stable for a long time.
- HEK293T cells were transfected with pNC1-FGF2. 48 hours after transfection, the cells were harvested and lysed to purify FGF2 expressed in the cells. FGF2 was purified by heparin-agarose affinity chromatography and then subjected to size exclusion chromatography. The eluted FGF2 was electrophoresed in 15% SDS-PAGE ( Figure 3A). The samples were lyophilized and reconstituted in 0.1x PBS. In order to check the purity of FGF2, silver staining was performed after electrophoresis and showed very high purity, only 1 weak band was found above the purified FGF2 ( Figure 3B). The yield is also satisfactory.
- FGF2 expressed by HEK293T has biological activity
- FGF2 has been shown to stimulate cell growth in many cell types. After confirming the primary structure of the purified FGF2, we treated the cultured C2C12 with 1ng/mL purified and commercially available FGF2, and PBS was used as a control. The viability of the cultured cells was then measured by the MTT assay (Figure 4A). Both FGF2 samples can stimulate the proliferation of C2C12 cells. Purified FGF2 showed similar or even higher biological activity than commercial FGF2, but the difference was not significant. FGF2 also exhibits neurotrophic activity in neuronal cell lines. Therefore, the neurotrophic activity of purified FGF2 was tested in PC12 cells. After 3 days of FGF2 treatment, prolonged neurite outgrowth was observed, which was not present in the PBS control group ( Figure 4B). The data confirms that the purified FGF2 has biological activity and has similar activity to commercial FGF2.
- the recombinant protein cascade containing 6x histidine-tagged DnaE-FGF2 was cloned into the pNC1 vector ( Figure 1C), and then transfected into HEK293T cells.
- the medium was collected and rotated at 2,000 g for 10 minutes to remove cell debris, and filtered through a 0.45 ⁇ m filter.
- the protein was purified by Ni-NTA affinity chromatography. FGF2 excision was performed on Ni-NTA column with lysis buffer (50mM Tris-HCl pH 6.2, 10mM EDTA, 200mM NaCl) at different time points at 22°C.
- FGF2 is a very valuable protein with a wide range of potency, including angiogenesis, neurogenesis and wound healing.
- One of the reasons hindering the research on the use of FGF2 in medicine is the high cost of purification and biological activity of FGF2.
- HEK 293T cells we successfully expressed and purified human FGF2 ( Figure 3). The silver-stained gel proved very satisfactory in purity.
- purified FGF2 exhibited the same mitogenic or neurotrophic activity as commercially available FGF2. This simple protocol allows for the production of human exogenous FGF2 on a laboratory scale in a mammalian system, and can be easily scaled up for large-scale production.
- endotoxin triggers the body's immune response and may lead to septic shock.
- kits and protocols for removing endotoxins endotoxin contamination is usually unavoidable.
- using mammalian cells can easily overcome the above-mentioned problems.
- DnaE is a fast-cutting intein in HEK293T cells.
- intein the primary structure (Table 3) and biological activity ( Figure 6) of excised FGF2 are the same as its natural counterpart.
- the medical applications of purified FGF2 will be extensive.
- the plasmid vector pNC1 can be used to express a variety of valuable proteins.
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
一种用于表达FGF-2的DNA序列,其中,该序列为SEQ ID NO.1或SEQ ID NO.2所示或者为其互补序列。还提供了一种构建体、其制备方法及应用,其中该构建体包括质粒载体EcoRI和NotI限制性酶切位点之间的如SEQ ID NO.1或SEQ ID NO.2所示DNA序列或者为其互补序列。还提供了一种制备FGF-2的方法。本发明提供的用于表达FGF-2的DNA序列及其构建体能显著提高具有生物活性FGF-2的表达,从而降低制备和纯化FGF-2的成本。
Description
本发明涉及一种用于表达FGF-2的DNA序列、含有所述DNA序列的构建体,以及该构建体的制备方法、该构建体在表达FGF-2中的用途,以及一种制备FGF-2的方法。
FGF2是制药和保健行业中非常有价值的蛋白质。FGF2是治疗神经退行性疾病,心脏病,血管生成,难以愈合的伤口和骨折的有效治疗性蛋白质;它还在干细胞的大规模生产中发挥重要作用。
由于制备成本低,复制率高,生产率高,大肠杆菌长期用于纯化重组蛋白[1-4]。然而,由于外源蛋白质引起的毒性,缺乏正确的蛋白质折叠和翻译后修饰,使用原核表达***在纯化哺乳动物蛋白质时经常遇到障碍[5,6]。多年来,科学家一直在使用真核宿主如酵母和昆虫细胞来克服这些局限[7]。尽管如此,使用人类来源的培养细胞进行人类来源的蛋白质表达似乎是直观的。实际上,出现了使用哺乳动物细胞产生重组蛋白的上升趋势[8,9]。
目前,FGF2制备和纯化最广泛使用的宿主***之一是细菌。然而,简单的原核生物缺乏必要的翻译后修饰,包括剪接,糖基化和二硫键,用于纯化的蛋白质的活性和溶解度。纯化蛋白质的天然折叠对其功能和溶解性至关重要。当在细菌***中表达真核蛋白时经常发现蛋白质聚集或包涵体。包括在较低温度下诱导蛋白质或使蛋白质聚集体变性和复性的方法并不总是产生良好的产率。因此,制备和纯化具有生物活性的FGF2极其昂贵(4000美元/毫克),这阻碍了其广泛的应用。
将多组氨酸(His-标签),谷胱甘肽S-转移酶(GST)或钙调蛋 白结合肽(CBP)等亲和标签融合到目标蛋白质是蛋白质纯化的常用策略[10]。标签提供了对细胞粗裂解物简单,简化的纯化方案。尽管可以在纯化后除去标签,但是通常在切割的蛋白质衍生物上留下一些额外的氨基酸。这些额外的氨基酸虽然很少,但可以改变折叠,从而改变其天然形式[11-14]。具有额外和不需要的氨基酸的重组蛋白质可能导致不可逆的有害作用,结合脱靶受体,并可能导致癌症[15,16]和过敏[16,17]。最近,许多研究小组报道了使用蛋白质内含子或内含肽来促进未标记蛋白的纯化[18-21]。内含肽是天然存在的蛋白质中的自切割元件。发现第一个鉴定的内含肽,VMA基因编码血管ATP酶,其中内含肽被切割,然后连接N末端和C末端外显肽以产生功能性全长蛋白[19-24]。现在在许多生物体中已经发现了400多个内含肽,科学家们已经在蛋白质纯化领域利用了这种自我切除特性。当设计适当时,内含肽的位点特异地、精确切割可以产生人类相同的蛋白质产物[23,25]。
发明内容
为了克服现有技术的缺陷,本发明一方面提供了一种用于表达FGF-2的DNA序列,其特征在于,该序列为SEQ ID NO.1所示:CCGGCTCTCCCAGAGGATGGCGGCTCAGGAGCCTTTCCACCAGGACACTTCAAAGATCCGAAGAGGCTTTACTGCAAGAATGGTGGATTTTTCCTCCGCATCCATCCAGACGGTCGGGTGGACGGCGTACGGGAGAAATCCGATCCGCATATAAAGCTGCAGCTGCAAGCTGAAGAACGAGGGGTGGTTAGCATAAAGGGCGTGTGTGCTAATAGGTACCTTGCCATGAAAGAAGACGGACGGCTCCTCGCTTCTAAGTGCGTGACCGACGAGTGCTTCTTCTTTGAGCGGCTAGAGTCAAACAATTATAACACCTATAGGTCAAGAAAGTATACGAGCTGGTACGTTGCCCTTAAGCGGACCGGCCAGTACAAGCTTGGTAGCAAAACAGGCCCTGGCCAGAAGGCTATCCTCTTCCTCCCTATGAGTGCCAAGTCTTAATAATAA,或者为其互补序列。
本发明还提供了另一种用于表达FGF-2的DNA序列,其特征在于,该序列如SEQ ID NO.2所示:
CATCATCACCATCACCACGCCGAGTACTACGAGACCGAGATCCTGACCGTGGAGTACGGCCTGCTGCCCATCGGCAAGATCGTGGAGAAGAGGATCGAGTGCACCGTGTACAGCGTGGACAACAACGGCAACATCTACACCCAGCCCGTGGCCCAGTGGCACGACAGGGGCGAGCAGGAGGTGTTCGAGTACTGCCTGGAGGACGGCAGCCTGATCAGGGCCACCAAGGACCACAAGTTCATGACCGTGGACGGCCAGATGCTGCCCATCGACGAGATCTTCGAGAGGGAGCTGGACCTGATGAGGGTGGACAACCTGCCCAACGCCGAGTACTACGAGACCGAGATCCTGACCGTGGAGTACGGCCTGCTGCCCATCGGCAAGATCGTGGAGAAGAGGATCGAGTGCACCGTGTACAGCGTGGACAACAACGGCAACATCTACACCCAGCCCGTGGCCCAGTGGCACGACAGGGGCGAGCAGGAGGTGTTCGAGTACTGCCTGGAGGACGGCAGCCTGATCAGGGCCACCAAGGACCACAAGTTCATGACCGTGGACGGCCAGATGCTGCCCATCGACGAGATCTTCGAGAGGGAGCTGGACCTGATGAGGGTGGACAACCTGCCCAACCCGGCTCTCCCAGAGGATGGCGGCTCAGGAGCCTTTCCACCAGGACACTTCAAAGATCCGAAGAGGCTTTACTGCAAGAATGGTGGATTTTTCCTCCGCATCCATCCAGACGGTCGGGTGGACGGCGTACGGGAGAAATCCGATCCGCATATAAAGCTGCAGCTGCAAGCTGAAGAACGAGGGGTGGTTAGCATAAAGGGCGTGTGTGCTAATAGGTACCTTGCCATGAAAGAAGACGGACGGCTCCTCGCTTCTAAGTGCGTGACCGACGAGTGCTTCTTCTTTGAGCGGCTAGAGTCAAACAATTATAACACCTATAGGTCAAGAAAGTATACGAGCTGGTACGTTGCCCTTAAGCGGACCGGCCAGTACAAGCTTGGTAGCAAAACAGGCCCTGGCCAGAAGGCTATCCTCTTCCTCCCTATGAGTGCCAAGTCTTAATAATAA,或者为其互补序列。
另一方面,本发明提供了一种构建体,其特征在于,该构建体 包括质粒载体EcoRI和NotI限制性酶切位点之间的如SEQ ID NO.1所示DNA序列或者为其互补序列,或者该构建体包括质粒载体EcoRI和NotI限制性酶切位点之间的如SEQ ID NO.2所示DNA序列或者为其互补序列。
另一方面,本发明提供了一种构建体的制备方法,其特征在于,所述方法包括以下步骤:
a.合成含有EcoRI和NotI限制性酶切位点的如SEQ ID NO.1所示的DNA序列或其互补序列;或者合成含有EcoRI和NotI限制性酶切位点的如SEQ ID NO.2所示的DNA序列或其互补序列;
b.用EcoRI和NotI消化质粒载体,回收大片段;
c.采用连接酶连接步骤a获得的DNA序列或者其互补序列与步骤b获得的大片段。
d.将步骤c获得的连接产物转染细胞,筛选阳性克隆,提取质粒。
另一方面,本发明提供了本发明提供的构建体在表达FGF-2中的应用。
另一方面,本发明提供了一种制备FGF-2的方法,其特征在于,该方法包括:
a.培养细胞:培养哺乳动物细胞;
b.转染:对a步骤获得的哺乳动物细胞进行转染如上所述的构建体;
c.纯化:通过亲和层析,纯化FGF-2。
本发明提供的用于表达FGF-2的DNA序列及其构建体能够显著提高FGF-2的表达水平,而且表达的FGF-2基本上是可溶的,其生物活性与商购的FGF-2的生物活性相当。因此,采用本发明提供的构建体表达FGF-2,能显著提高具有生物活性FGF-2的表达,从而能大大降低制备和纯化FGF-2的成本。
此处所说明的附图用来提供对本发明的进一步理解,构成本申请的一部分,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1是FGF2构建体和his-DnaE-FGF2构建体的示意图。(A)合成fgf2基因的成熟功能片段的DNA,并克隆到在CMV增强子/启动子的控制下的pcDNA 3.1(+)载体中,形成pcDNA3.1-FGF2构建体。(B1)合成NF-κB结合位点和CREB结合位点并克隆在CMV启动子后,形成pNC1载体;(B2)然后将fgf2基因的成熟功能片段的DNA克隆到pNC1载体中,形成pNC1-FGF2构建体。(C)通过融合PCR将6xhis标签和Npu DnaE内含肽基因与fgf2融合,并克隆到pNC1载体中,形成pNC1-6xhis-DnaE-FGF2构建体。
图2是成熟功能片段FGF2蛋白在HEK293T细胞中的表达。(A)将pcDNA3.1-FGF2和pNC1-FGF2构建体转染到HEK293T细胞中,并在指定的时间点通过蛋白质印迹分析全细胞裂解物。(B)为(A)相对β-肌动蛋白标准化的光密度分析。pNC1-FGF2构建体在24小时,36小时和48小时显示出比pcDNA3.1-FGF2构建体更高的表达水平(**p<0.01,***p<0.001;在每个时间点n=5)。(C)通过蛋白质印迹分析来自细胞裂解物的上清液(S)和沉淀(P)。商购FGF2(+ve)作为阳性对照。对于pcDNA3.1-FGF2和pNC1-FGF2构建体,HEK293T表达的FGF2都是相当可溶的。
图3是在HEK293T细胞中表达的FGF2的纯化。(A)将全细胞裂解物和来自尺寸排阻色谱样品的洗脱物进行SDS-PAGE并用考马斯亮蓝染色。(B)将来自(A)的样品冻干并在0.1x PBS中重构,然后在SDS-PAGE后通过银染色分析。在纯化的FGF2中仅观察到1个弱带(由箭头指示)。
图4是纯化的FGF2的生物测定。(A)MTT测定以测量FGF2相对于PBS对照对于C2C12细胞的细胞活力的影响。在指定的时间点可以观察到在培养基中1ng/mL的商购和纯化的FGF2都能够诱导细胞生长(n=3)。(B)将浓度为2ng/mL的商购和纯化的FGF2 加入培养基中3天。在第0天和第3天拍摄相差显微照片。在FGF2处理3天后观察到PC12细胞中的延长的神经突生长(用箭头表示)。
图5是成熟功能片段FGF2蛋白在HEK293T细胞中的表达以及通过内含肽辅助切割纯化FGF2。将pNC1-6xhis-DnaE-FGF2构建体转染到HEK293T细胞中,收集培养基并以2,000g旋转10分钟以除去细胞碎片,并通过0.45μm过滤器过滤。通过Ni-NTA亲和层析纯化蛋白质。在切割缓冲液中诱导DnaE内含肽的切割。从诱导后2小时开始,将全长his-DnaE-FGF2蛋白(由*表示的条带)切割成FGF2(用#表示的条带)。诱导后6小时后裂解完成。
图6是纯化的和内切蛋白切割的FGF2的生物测定。(A)MTT测定以测量FGF2相对于PBS对照对于C2C12细胞的细胞活力的影响。在指定的时间点观察到在培养基中1ng/mL的纯化和内切蛋白切割的FGF2均能够诱导细胞生长(n=3)。(B)将2ng/mL的纯化和内含肽切割的FGF2加入培养基中3天。在第0天和第3天拍摄相差显微照片。在FGF2处理3天后观察到PC12细胞中的延长的神经突生长(用箭头表示)。
需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
本发明提供了一种用于表达FGF-2的DNA序列,其特征在于,该序列为SEQ ID NO.1所示:
CCGGCTCTCCCAGAGGATGGCGGCTCAGGAGCCTTTCCACCAGGACACTTCAAAGATCCGAAGAGGCTTTACTGCAAGAATGGTGGATTTTTCCTCCGCATCCATCCAGACGGTCGGGTGGACGGCGTACGGGAGAAATCCGATCCGCATATAAAGCTGCAGCTGCAAGCTGAAGAACGAGGGGTGGTTAGCATAAAGGGCGTGTGTGCTAATAGGTACCTTGCCATGAAAGAAGACGGACGGCTCCTCGCTTCTAAGTG CGTGACCGACGAGTGCTTCTTCTTTGAGCGGCTAGAGTCAAACAATTATAACACCTATAGGTCAAGAAAGTATACGAGCTGGTACGTTGCCCTTAAGCGGACCGGCCAGTACAAGCTTGGTAGCAAAACAGGCCCTGGCCAGAAGGCTATCCTCTTCCTCCCTATGAGTGCCAAGTCTTAATAATAA,或者为其互补序列。
本发明提还供了一种用于表达FGF-2的DNA序列,其特征在于,其特征在于,该序列为SEQ ID NO.2所示:
CATCATCACCATCACCACGCCGAGTACTACGAGACCGAGATCCTGACCGTGGAGTACGGCCTGCTGCCCATCGGCAAGATCGTGGAGAAGAGGATCGAGTGCACCGTGTACAGCGTGGACAACAACGGCAACATCTACACCCAGCCCGTGGCCCAGTGGCACGACAGGGGCGAGCAGGAGGTGTTCGAGTACTGCCTGGAGGACGGCAGCCTGATCAGGGCCACCAAGGACCACAAGTTCATGACCGTGGACGGCCAGATGCTGCCCATCGACGAGATCTTCGAGAGGGAGCTGGACCTGATGAGGGTGGACAACCTGCCCAACGCCGAGTACTACGAGACCGAGATCCTGACCGTGGAGTACGGCCTGCTGCCCATCGGCAAGATCGTGGAGAAGAGGATCGAGTGCACCGTGTACAGCGTGGACAACAACGGCAACATCTACACCCAGCCCGTGGCCCAGTGGCACGACAGGGGCGAGCAGGAGGTGTTCGAGTACTGCCTGGAGGACGGCAGCCTGATCAGGGCCACCAAGGACCACAAGTTCATGACCGTGGACGGCCAGATGCTGCCCATCGACGAGATCTTCGAGAGGGAGCTGGACCTGATGAGGGTGGACAACCTGCCCAACCCGGCTCTCCCAGAGGATGGCGGCTCAGGAGCCTTTCCACCAGGACACTTCAAAGATCCGAAGAGGCTTTACTGCAAGAATGGTGGATTTTTCCTCCGCATCCATCCAGACGGTCGGGTGGACGGCGTACGGGAGAAATCCGATCCGCATATAAAGCTGCAGCTGCAAGCTGAAGAACGAGGGGTGGTTAGCATAAAGGGCGTGTGTGCTAATAGGTACCTTGCCATGAAAGAAGACGGACGGCTCCTCGCTTCTAAGTGCGTGACCGACGAGTGCTTCTTCTTTGAGCGGCTAGAGTCAAACAATTATAACACCTAT AGGTCAAGAAAGTATACGAGCTGGTACGTTGCCCTTAAGCGGACCGGCCAGTACAAGCTTGGTAGCAAAACAGGCCCTGGCCAGAAGGCTATCCTCTTCCTCCCTATGAGTGCCAAGTCTTAATAATAA,或者为其互补序列。该DNA序列融合了Npu DnaE内含肽基因,以便于进行内含肽介导的分离纯化。
本发明还提供了一种构建体,其特征在于,该构建体包括质粒载体EcoRI和NotI限制性酶切位点之间的如SEQ ID NO.1所示DNA序列或者为其互补序列,或者该构建体包括质粒载体EcoRI和NotI限制性酶切位点之间的如SEQ ID NO.2所示DNA序列或者为其互补序列。
对于本发明的构建体,尽管对质粒没有特别的要求,但是为了提高FGF-2的表达水平,在一种优选的实施方式中,所述质粒载体为pcDNA 3.1(+)或者pNC1,更优选为pNC1。
为了进一步提高FGF-2的表达水平以及便于纯化,在一种优选的实施方式中,所述构建体包括质粒载体EcoRI和NotI限制性酶切位点之间的如SEQ ID NO.2所示DNA序列或者为其互补序列,所述质粒载体为pNC1。
本发明还提供了一种构建体的制备方法,其特征在于,所述方法包括以下步骤:
a.合成含有EcoRI和NotI限制性酶切位点的如SEQ ID NO.1所示的DNA序列或其互补序列;或者合成含有EcoRI和NotI限制性酶切位点的如SEQ ID NO.2所示的DNA序列或其互补序列;
b.用EcoRI和NotI消化质粒载体,回收大片段;
c.采用连接酶连接步骤a获得的DNA序列或者其互补序列与步骤b获得的大片段。
d.将步骤c获得的连接产物转染细胞,筛选阳性克隆,提取质粒。
在一种优选的实施方式中,所述质粒载体为pcDNA 3.1(+)或者pNC1,更优选为pNC1。
本发明还提供了所述构建体在表达FGF-2中的用途。
本发明还提供了一种制备FGF-2的方法,其特征在于,该方法包括:
a.培养细胞:培养哺乳动物细胞;
b.转染:对a步骤获得的哺乳动物细胞进行转染如上所述的构建体;
c.纯化:通过亲和层析,纯化FGF-2。
在一种优选的实施方式中,所述亲和层析通过肝素结合结构域进行。
在一种优选的实施方式中,在所述步骤b中,对a步骤获得的哺乳动物细胞进行转染如上所述的构建体;所述亲和层析为通过内含肽DnaE介导的亲和层析。
在本发明制备FGF-2的方法中,尽管对哺乳动物细胞没有特别限制,例如可以采用HEK293T、HeLa、C2C12等。但在一种优选的实施方式中,所述哺乳动物细胞为HEK 293T。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
实施例
材料和方法
化学品、质粒和抗体,除非另有说明,否则所有化学品均购自Sigma-Aldrich(St.Louis,MO)。
质粒载体pNC1的构建
如图1中的B1所示:
a.采用PCR(用GeneArt Strings基因合成服务(Thermo-Fisher Scientific,Waltham,MA))合成增强子序列,该增强子序列为含 有NF-κB结合位点的DNA序列和CREB结合位点的DNA序列的DNA序列;
b.用NheI和HindIII消化质粒载体pcDNA 3.1(+);
c.用NheI和HindIII消化步骤a合成的DNA序列;
d.采用连接酶连接步骤b获得的消化后的质粒载体pcDNA 3.1(+)与步骤c获得的消化后的DNA序列。
通过Sanger测序确认序列。
具体过程如下:
材料
pcDNA 3.1(+)购自美国Thermo Scientific公司,其载体图谱如图1A所示。限制性内切酶NheI和HindIII,以及T4 DNA Ligase购自NEB公司。质粒提取试剂盒和DNA片段回收试剂盒购自Thermo Scientific公司。
增强子PCR扩增
增强子的模板通过GeneArt string service(thermos scientific)合成,其DNA序列如SEQ ID NO.5所示:5’GGAAATCCCCGGAAATCCCCGTAAAATTTGCGTCAACACTGCTCAAC。采用的正向和反向引物如表1所示。在EP管中依次加入以下反应物:0.5μl的模板,1x缓冲液,1μM正向引物,1μM反向引物,0.5μl聚合酶,4μl dNTP,加ddH2O至50μl反应体系中进行反应。
反应条件:95℃、5min;95℃、15s;55℃、10s;72℃、10s;35个循环;72℃延伸10min,4℃终止反应。
将PCR扩增产物在3%琼脂糖凝胶中电泳进行结果检测,用DNA胶回收纯化试剂盒回收***条带。获得59bp的片段,与预期大小一致。
将上述PCR回收产物连接至质粒载体pcDNA 3.1(+)上
将上述PCR回收片段和质粒载体pcDNA 3.1(+)用NheI和HindIII酶切处理,然后用T4DNA连接酶进行连接,连接产物转化 至化学感受态细胞DH5a菌株中,涂布到氨苄抗性固体培养基平皿,挑取若干单克隆菌落,接种到氨苄抗性液体培养基中,恒温摇床37℃、250rpm振荡培养过夜。采用质粒提取试剂盒提取质粒,得到重组质粒载体pNC1。
重组质粒载体pNC1的鉴定
用NheI和HindIII酶切进行鉴定,酶切鉴定体系及反应条件如下表:
重组质粒DNA 5μL,10×缓冲液2μL,NheI 0.5μL,HindIII 0.5μL,ddH2O 11μL
总体积20μL。37℃,15min。
经酶切鉴定,获得产物与预期分析大小一致。
选取酶切验证正确的阳性克隆进行测序分析,结果经FinchTV解析及BLAST软件比对,以验证重组质粒载体的正确性。分析结果表明,增强子核苷酸序列完全正确,成功获得了重组质粒载体pNC1。
FGF2-pcDNA3.1(+)构建体、FGF2-pNC1构建体和his-DnaE-FGF2-pNC1构建体
没有前肽序列的fgf2基因(即fgf2基因的成熟功能片段)的DNA序列(氨基酸143-288,PRO_0000008933)被设计用于人类的密码子优化。
用GeneArt Strings基因合成服务(Thermo-Fisher Scientific,Waltham,MA)合成设计的fgf2基因,将合成的fgf2基因克隆到具有EcoRI和NotI位点的pcDNA3.1(+)和pNC1中,形成FGF2-pcDNA3.1(+)构建体和FGF2-pNC1构建体,如图1A,1B2所示。通过Sanger测序确认所有序列。
为了优化纯化,本发明使用6x his标签以及使用融合了fgf2基因的点形念珠藻(Nostoc punctiforme)PCC73102(Npu)的DNA聚合酶III(DnaE)内含肽来促进人成纤维细胞生长因子2(FGF2) 的纯化。
用GeneArt Strings基因合成服务(Thermo-Fisher Scientific,Waltham,MA)合成设计的fgf2和6x his-Npu DnaE内含肽基因。通过重叠PCR将6x his-Npu DnaE内含肽与fgf2融合。
为了进一步提高fgf2的表达,使用GeneArt Strings基因合成服务合成含有NF-κB结合位点和CREB结合位点的增强子序列,并克隆到具有NheI和HindIII位点的pcDNA 3.1(+)中(Thermo-Fisher Scientific,Waltham,MA))以形成表达质粒载体pNC1。将合成6xhis-DnaE-FGF2)克隆到具有EcoRI和NotI位点的pNC1中,形成his-DnaE-FGF2-pNC1构建体,如图1C所示。通过Sanger测序确认所有序列。用于蛋白质印迹的抗体:小鼠FGF-2(克隆C-2,Santa Cruz Biotechnology,Dallas,TX),小鼠抗β-肌动蛋白(Sigma,St.Louis,MO)。
FGF2-pcDNA3.1(+)构建体的构建具体过程如下:
fgf2基因PCR扩增
fgf2的模板通过GeneArt string service(thermos scientific)合成,其DNA序列如SEQ ID NO.1所示
CCGGCTCTCCCAGAGGATGGCGGCTCAGGAGCCTTTCCACCAGGACACTTCAAAGATCCGAAGAGGCTTTACTGCAAGAATGGTGGATTTTTCCTCCGCATCCATCCAGACGGTCGGGTGGACGGCGTACGGGAGAAATCCGATCCGCATATAAAGCTGCAGCTGCAAGCTGAAGAACGAGGGGTGGTTAGCATAAAGGGCGTGTGTGCTAATAGGTACCTTGCCATGAAAGAAGACGGACGGCTCCTCGCTTCTAAGTGCGTGACCGACGAGTGCTTCTTCTTTGAGCGGCTAGAGTCAAACAATTATAACACCTATAGGTCAAGAAAGTATACGAGCTGGTACGTTGCCCTTAAGCGGACCGGCCAGTACAAGCTTGGTAGCAAAACAGGCCCTGGCCAGAAGGCTATCCTCTTCCTCCCTATGAGTGCCAAGTCTTAATAATAA。采用的正向和反向引物如表1所示。在EP管中依次加入以下反应物:0.5μl的模板,1x缓冲液,1μM正 向引物,1μM反向引物,0.5μl聚合酶,4μl dNTP,加ddH2O至50μl反应体系中进行反应。
反应条件:95℃、5min;95℃、15s;55℃、10s;72℃、30min;35个循环;72℃延伸10min,4℃终止反应。
将PCR扩增产物在1%琼脂糖凝胶中电泳进行结果检测,用DNA胶回收纯化试剂盒回收***条带。获得460bp的片段,与预期大小一致。
将上述PCR回收产物连接至质粒载体pcDNA 3.1(+)上
将上述PCR回收片段和质粒载体pcDNA 3.1(+)用EcoRI和NotI酶切处理,然后用T4DNA连接酶进行连接,连接产物转化至化学感受态细胞DH5a菌株中,涂布到氨苄抗性固体培养基平皿,挑取若干单克隆菌落,接种到氨苄抗性液体培养基中,恒温摇床37℃、250rpm振荡培养过夜。采用质粒提取试剂盒提取质粒,得到FGF2-pcDNA3.1(+)构建体。
FGF2-pcDNA3.1(+)构建体的鉴定
用EcoRI和NotI酶切进行鉴定,酶切鉴定体系及反应条件如下表:
FGF2-pcDNA3.1(+)构建体5μL,10×缓冲液2μL,EcoRI 0.5μL,NotI 0.5μL,ddH2O 11μL总体积20μL。37℃,15min。
经酶切鉴定,获得产物与预期分析大小一致。
选取酶切验证正确的阳性克隆进行测序分析,结果经FinchTV解析及BLAST软件比对,以验证FGF2-pcDNA3.1(+)构建体的正确性。分析结果表明,fgf2核苷酸序列完全正确,成功获得了FGF2-pcDNA3.1(+)构建体。
FGF2-pNC1构建体的构建具体过程如下:
fgf2基因PCR扩增
fgf2的模板通过GeneArt string service(thermos scientific)合成, 其DNA序列如SEQ ID NO.1所示:
CCGGCTCTCCCAGAGGATGGCGGCTCAGGAGCCTTTCCACCAGGACACTTCAAAGATCCGAAGAGGCTTTACTGCAAGAATGGTGGATTTTTCCTCCGCATCCATCCAGACGGTCGGGTGGACGGCGTACGGGAGAAATCCGATCCGCATATAAAGCTGCAGCTGCAAGCTGAAGAACGAGGGGTGGTTAGCATAAAGGGCGTGTGTGCTAATAGGTACCTTGCCATGAAAGAAGACGGACGGCTCCTCGCTTCTAAGTGCGTGACCGACGAGTGCTTCTTCTTTGAGCGGCTAGAGTCAAACAATTATAACACCTATAGGTCAAGAAAGTATACGAGCTGGTACGTTGCCCTTAAGCGGACCGGCCAGTACAAGCTTGGTAGCAAAACAGGCCCTGGCCAGAAGGCTATCCTCTTCCTCCCTATGAGTGCCAAGTCTTAATAATAA。采用的正向和反向引物如表1所示。在EP管中依次加入以下反应物:0.5μl的模板,1x缓冲液,1μM正向引物,1μM反向引物,0.5μl聚合酶,4μl dNTP,加ddH2O至50μl反应体系中进行反应。
反应条件:95℃、5min;95℃、15s;55℃、10s;72℃、30min;35个循环;72℃延伸10min,4℃终止反应。将PCR扩增产物在1%琼脂糖凝胶中电泳进行结果检测,用DNA胶回收纯化试剂盒回收***条带。获得460bp的片段,与预期大小一致。
将上述PCR回收产物连接至质粒载体pNC1上
将上述PCR回收片段和质粒载体pNC1用EcoRI和NotI酶切处理,然后用T4DNA连接酶进行连接,连接产物转化至化学感受态细胞DH5a菌株中,涂布到氨苄抗性固体培养基平皿,挑取若干单克隆菌落,接种到氨苄抗性液体培养基中,恒温摇床37℃、250rpm振荡培养过夜。采用质粒提取试剂盒提取质粒,得到pNC1构建体。
FGF2-pNC1构建体的鉴定
用EcoRI和NotI酶切进行鉴定,酶切鉴定体系及反应条件如下表:
FGF2-pNC1构建体5μL,10×缓冲液2μL,EcoRI 0.5 μL,NotI 0.5μL,ddH
2O 11μL总体积20μL。37℃,15min。经酶切鉴定,获得产物与预期分析大小一致。
选取酶切验证正确的阳性克隆进行测序分析,结果经FinchTV解析及BLAST软件比对,以验证FGF2-pNC1构建体的正确性。分析结果表明,fgf2核苷酸序列完全正确,成功获得了FGF2-pNC1构建体。
his-DnaE-FGF2-pNC1构建体的构建具体过程如下:
his-DnaE-FGF2的融合PCR扩增
his-DnaE-FGF2的模板通过GeneArt string service(thermos scientific)合成,其DNA序列如SEQ ID NO.2所示:
CATCATCACCATCACCACGCCGAGTACTACGAGACCGAGATCCTGACCGTGGAGTACGGCCTGCTGCCCATCGGCAAGATCGTGGAGAAGAGGATCGAGTGCACCGTGTACAGCGTGGACAACAACGGCAACATCTACACCCAGCCCGTGGCCCAGTGGCACGACAGGGGCGAGCAGGAGGTGTTCGAGTACTGCCTGGAGGACGGCAGCCTGATCAGGGCCACCAAGGACCACAAGTTCATGACCGTGGACGGCCAGATGCTGCCCATCGACGAGATCTTCGAGAGGGAGCTGGACCTGATGAGGGTGGACAACCTGCCCAACGCCGAGTACTACGAGACCGAGATCCTGACCGTGGAGTACGGCCTGCTGCCCATCGGCAAGATCGTGGAGAAGAGGATCGAGTGCACCGTGTACAGCGTGGACAACAACGGCAACATCTACACCCAGCCCGTGGCCCAGTGGCACGACAGGGGCGAGCAGGAGGTGTTCGAGTACTGCCTGGAGGACGGCAGCCTGATCAGGGCCACCAAGGACCACAAGTTCATGACCGTGGACGGCCAGATGCTGCCCATCGACGAGATCTTCGAGAGGGAGCTGGACCTGATGAGGGTGGACAACCTGCCCAACCCGGCTCTCCCAGAGGATGGCGGCTCAGGAGCCTTTCCACCAGGACACTTCAAAGATCCGAAGAGGCTTTACTGCAAGAATGGTGGATTTTTCCTCCGCATCCATCCAGACGGTCGGGTGGACGGCGTACGGGAGAAATCCGATC CGCATATAAAGCTGCAGCTGCAAGCTGAAGAACGAGGGGTGGTTAGCATAAAGGGCGTGTGTGCTAATAGGTACCTTGCCATGAAAGAAGACGGACGGCTCCTCGCTTCTAAGTGCGTGACCGACGAGTGCTTCTTCTTTGAGCGGCTAGAGTCAAACAATTATAACACCTATAGGTCAAGAAAGTATACGAGCTGGTACGTTGCCCTTAAGCGGACCGGCCAGTACAAGCTTGGTAGCAAAACAGGCCCTGGCCAGAAGGCTATCCTCTTCCTCCCTATGAGTGCCAAGTCTTAATAATAA。以His-DnaE正向引物和FGF2反向引物,采用的正向和反向引物如表1所示。在EP管中依次加入以下反应物:0.5μl的模板,1x缓冲液,1μM正向引物,1μM反向引物,0.5μl聚合酶,4μl dNTP,加ddH2O至50μl反应体系中进行反应。
反应条件:95℃、5min;95℃、15s;55℃、10s;72℃、30min;35个循环;72℃延伸10min,4℃终止反应。将PCR扩增产物在1%琼脂糖凝胶中电泳进行结果检测,用DNA胶回收纯化试剂盒回收GFP条带。获得1097bp的片段,与预期大小一致。
将上述PCR回收产物连接至质粒载体his-DnaE-FGF2上
将上述PCR回收片段和质粒载体his-DnaE-FGF2用EcoRI和NotI酶切处理,然后用T4DNA连接酶进行连接,连接产物转化至化学感受态细胞DH5a菌株中,涂布到氨苄抗性固体培养基平皿,挑取若干单克隆菌落,接种到氨苄抗性液体培养基中,恒温摇床37℃、250rpm振荡培养过夜。采用质粒提取试剂盒提取质粒,得到his-DnaE-FGF2构建体。
his-DnaE-FGF2构建体的鉴定
用EcoRI和NotI酶切进行鉴定,酶切鉴定体系及反应条件如下表:
his-DnaE-FGF2构建体5μL,10×缓冲液2μL,EcoRI 0.5μL,NotI 0.5μL,ddH2O 11μL总体积20μL。37℃,15min。
经酶切鉴定,获得产物与预期分析大小一致。
选取酶切验证正确的阳性克隆进行测序分析,结果经FinchTV解析及BLAST软件比对,以验证his-DnaE-FGF2构建体的正确性。分析结果表明,his-DnaE-fgf2核苷酸序列完全正确,成功获得了his-DnaE-FGF2构建体。
表1
PCR合成的含有NheI和HindIII限制性酶切位点增强子的DNA为SEQ ID NO.11(即为含有NheI和HindIII限制性酶切位点SEQ ID NO.5):
PCR合成的含有EcoRI和NotI限制性酶切位点的FGF2的DNA序列为SEQ ID NO.12(即为含有EcoRI和NotI限制性酶切位点SEQ ID NO.1):
PCR合成的带有EcoRI和NotI限制性酶切位点的his-DnaE-FGF2的DNA序列为SEQ ID NO.13(即为含有EcoRI和NotI限制性酶切位点SEQ ID NO.2):
细胞培养和转染
将HEK 293T和C2C12细胞维持在含有10体积%FBS和1体积%青霉素链霉素溶液(Thermo-Fisher Scientific,Waltham,MA)的DMEM中,环境中温度为37℃,含有5%CO2。将PC12细胞维持在含有10体积%HS,5体积%FBS和1体积%青霉素链霉素溶液的DMEM中,环境中温度为37℃,含有5%CO2。根据制造商的说明,用Lipofectamine 2000(Thermo-Fisher Scientific,Waltham,MA)将pcDNA3.1-FGF2,pNC1-FGF2和pNC1-6xhis-DnaE-FGF2构建体转染到HEK 293T中。
FGF2的纯化
收集培养基并以2,000g旋转10分钟以除去细胞碎片,并通过0.45μm过滤器过滤。然后将滤液通过用50mM Tris-HCl(pH7.5)平衡的预装的肝素-琼脂糖柱(BioRad Laboratories,Hercules,CA)。用50mM Tris-HCl(pH 7.5),0.2M NaCl彻底洗涤柱子。用0.3M至3M(4-5床体积用于梯度)的NaCl梯度洗脱FGF2。洗脱后,使蛋白质通过用50mM Tris-HCl(pH7.5)平衡的预装的Sephadex G25柱,并用相同的缓冲液洗脱。
对于细胞内表达的FGF2,将细胞用冰冷的TBS洗涤三次,然后在裂解缓冲液(提供有cOmpleteTM蛋白酶抑制剂混合物(cOmpleteTM Protease Inhibitor Cocktail)的TBS)中超声处理。然后在培养基中纯化FGF2。
为了获得内含肽切除的FGF2,通过预先包装的Ni-NTA柱纯化 组氨酸标记的DnaE-FGF2。洗涤后,将柱在50mM Tris-HCl(pH6.2),10mM EDTA,200mM NaCl中于22℃温育不同的持续时间,分别为0、1、2、4、6、10小时,以诱导DnaE内含肽的C末端切除。
蛋白质分析
在15体积%Tris-甘氨酸SDS-PAGE中分离蛋白质。凝胶用银染以获得纯化的蛋白质。在凝胶上,切下对应于FGF2的条带,洗涤并与1μg胰蛋白酶在50mM NH 4 HCO 3中于4℃温育过夜。通过与Accela HPLE***结合的LTQ Velos线性离子阱质谱仪(LTQ Velos Linear Ion Trap Mass Spectrometer)(Thermo Fisher Scienctific,San Jose)分析水解的样品。获得完整的MS扫描(300-2000m/z)并用Mascot搜索引擎(Matric Science,Boston,MA)鉴定肽序列。对于细胞裂解物,将样品点样或转移到0.2μmNC膜(BioRad Laboratories,Hercules,CA)上,然后用抗体印迹(小鼠FGF-2(克隆C-2,Santa Cruz Biotechnology,Dallas,TX),小鼠抗β-肌动蛋白(Sigma,St.Louis,MO))。
FGF2的生物学测定
为了监测FGF2对细胞增殖的影响,如前所述进行MTT测定。简而言之,将C2C12细胞接种在96孔板上,并提供DMEM+0.5体积%FBS和1ng/mL的商购FGF2,纯化的FGF2和内切肽切除的FGF2。通过加入MTT至终浓度1mg/mL测定细胞的活性,并在37℃下孵育6小时。然后用DMSO替换培养基,并在微量板读数器中在540nm处测量吸光度。
为了检测FGF2的神经营养作用,将PC12细胞与提供有2ng/mL的FGF2的培养基一起培养3天。观察细胞的形态,并通过相差光学显微镜拍摄图像。
结果
构建表达人相同的外源FGF2的质粒
全长FGF2蛋白由288个氨基酸组成,其中氨基酸1-142被切割并除去以产生功能性FGF2。因此,氨基酸143-288的蛋白质序列经过密码子优化,用于智人(Homo sapiens)。为了探索蛋白质最大化表达的可行性,将NF-κB结合位点和CREB结合位点增强子序列克隆到含有天然CMV立即早期启动子/增强子序列的pcDNA3.1载体中。然后将合成的fgf2基因克隆到pcDNA3.1和pNC1载体中(图1),用于比较两个克隆在哺乳动物细胞中的表达。实验表明pNC1载体大大提高了FGF2在哺乳动物细胞中的表达水平,如图2所示。新霉素抗性基因还通过G418硫酸盐提供选择标记。因此可以选择稳定转染的细胞用于纯化人相同的外源FGF2。
可溶性FGF2在HEK 293T细胞中的表达
选择HEK293T细胞用于纯化,因为其易于转染。SV40大T抗原还提供细胞复制转染质粒的能力,其含有SV40复制起点。此外,CMV启动子是最强的启动子之一,并且在HEK293T细胞中具有组成型活性。虽然HEK293T细胞是贴壁细胞,但它适用于悬浮培养是通用的,因此有利于扩大FGF2的产生。将pcDNA3.1-FGF2 pNC1-FGF2和pNC1-6xhis-DnaE-FGF2构建体瞬时转染至HEK293T细胞并监测表达。实验表明:pNC1载体大大提高了FGF2的表达水平(图2A,2B)。FGF2的表达在转染后48小时达到峰值并且在较长时间点保持稳定状态。
使用细菌***进行蛋白质制备和纯化的缺点之一是形成不溶性聚集体。为了测试在HEK293T细胞中表达的FGF2的溶解度,从细胞裂解中收获沉淀级分。将不溶性级分在Laemmili样品缓冲液中超声处理,然后在SDS-PAGE中电泳。裂解物的蛋白质印迹显示绝大部分FGF2是可溶的(图2C)。与FGF2标准相比,在pcDNA3.1-FGF2中表达的FGF2约为20μg/mL,对于在pNC1-FGF2中表达的FGF2约为50μg/mL。
FGF2的纯化和蛋白质测序
为了使纯化的FGF2的产量最大化,用pNC1-FGF2转染HEK293T细胞。转染后48小时,收获并裂解细胞以纯化细胞内表达的FGF2。通过肝素-琼脂糖亲和层析纯化FGF2,然后进行尺寸排阻层析。洗脱的FGF2在15%SDS-PAGE中电泳(图3A)。将样品冻干并在0.1x PBS中重构。为了检查FGF2的纯度,在电泳后进行银染色并且显示出非常高的纯度,在纯化的FGF2上方仅发现1个弱条带(图3B)。产量也令人满意。为了鉴定纯化的FGF2,切下SDS-PAGE上的条带,溶解并在胰蛋白酶消化后通过LC-MS进行分析。测序结果显示纯化的FGF2的一级结构与成熟的外源人FGF2相同(表2)。因此,我们预期纯化的FGF2表现出与人天然对应物相同的性质,活性和功能。
HEK293T表达的FGF2具有生物学活性
已经显示FGF2在许多细胞类型中刺激细胞生长。在确认纯化的FGF2的一级结构后,我们用1ng/mL的纯化和商购FGF2处理培养的C2C12,PBS用作对照。然后通过MTT测定法测量培养细胞的活力(图4A)。两种FGF2样品都能够刺激C2C12细胞的增殖。纯化的FGF2显示出与商购FGF2相似甚至更高的生物活性,但差异不显着。FGF2还在神经元细胞系中表现出神经营养活性。因此,在PC12细胞中测试了纯化的FGF2的神经营养活性。在FGF2处理3天后,观察到延长的神经突生长,其在PBS对照组中不存在(图4B)。数据证实纯化的FGF2具有生物学活性,并具有与商购FGF2相似的活性。
在HEK293T细胞中内含肽介导的纯化
将含有6x组氨酸标记的DnaE-FGF2的重组蛋白质级联克隆到pNC1载体中(图1C),然后转染到HEK293T细胞。收集培养基并以2,000g旋转10分钟以除去细胞碎片,并通过0.45μm过滤器过滤。通过Ni-NTA亲和层析纯化蛋白质。用裂解缓冲液(50mM Tris-HCl pH 6.2,10mM EDTA,200mM NaCl)在Ni-NTA柱上进行FGF2切除,在22℃下进行不同的时间点。将珠子和上清液在2x Laemmili样品缓冲液中煮沸以洗脱珠子结合的蛋白质,然后进行SDS-PAGE(图5)。实验结果表明,内含肽的完全切割需要至少5小时的孵育。通过LC-MS测定切除的蛋白质的鉴定,发现切除的FGF2的一级序列与成熟的人FGF2相同(表3)。还测试了切除的FGF2对C2C12细胞的生物活性,并且内切蛋白切除的FGF2的性能也与纯化的FGF2相同(图6A,6B)。
表2
液相色谱-串联质谱法分析纯化的FGF2
a在胰蛋白酶部分消化纯化的FGF2后,通过Mascot搜索引擎鉴定N-末端和C-末端序列
b肽的理论质荷比
c肽的实验质荷比
表3
用液相色谱-串联质谱法分析内含肽-切除的FGF2
a在胰蛋白酶部分消化纯化的FGF2后,通过Mascot搜索引擎鉴定N-末端和C-末端序列
b肽的理论质荷比
c肽的实验质荷比
FGF2是一种非常有价值的蛋白质,具有广泛的效力,包括血管生成,神经发生和伤口愈合。阻碍FGF2在医学上使用的研究的原因之一是纯化和生物活性FGF2的高成本。使用HEK 293T细胞,我们成功表达并纯化了人源FGF2(图3)。银染的凝胶证实纯度非常令人满意。在C2C12和PC12细胞培养物中,纯化的FGF2表现出与市售FGF2相同的促有丝***或神经营养活性。这种简单的方案允许在哺乳动物***中实验室规模生产人外源性FGF2,并且可以容易地按比例放大以进行大规模生产。
用于FGF2纯化的最广泛使用的宿主***之一是细菌。然而,简单的原核生物缺乏必要的翻译后修饰,包括剪接,糖基化和二硫键,用于纯化的蛋白质的活性和溶解度。纯化蛋白质的天然折叠对其功能和溶解性至关重要。当在细菌***中表达真核蛋白时经常发现蛋白质聚集或包涵体。包括在较低温度下诱导蛋白质或使蛋白质聚集体变性和复性的方法并不总是产生良好的产率。使用细菌***的另一个问题是内毒素。脂多糖或内毒素通常存在于大肠杆菌中。内毒素的存在会引发人体免疫反应,并可能导致感染性休克。虽然有去除内毒素的商购试剂盒和方案,但内毒素的污染通常是不可避免的。然而,使用哺乳动物细胞,可以容易地克服上述问题。
使用哺乳动物细胞表达蛋白质,我们能够纯化功能性,正确折叠和修饰的蛋白质(表2,图3、图4)。因此,我们是第一次在哺乳动物细胞中成功纯化无标记的人成熟外源FGF2。
在本发明中,我们已经证明DnaE是HEK293T细胞中的快速切割内含肽。通过使用这种内含肽,我们已经证实切除的FGF2的一级结构(表3)和生物活性(图6)与其天然对应物相同。纯化的FGF2的医学应用将是广泛的。此外,质粒载体pNC1可用于表达多种有价值的蛋白质。
参考文献
[1]N.Ferrer-Miralles,J.Domingo-Espín,J.L.Corchero,E.Vázquez,and A.Villaverde,“Microbial factories for recombinant pharmaceuticals,”Microbial Cell Factories,vol.8,no.1,p.17,Mar.2009.
[2]J.H.Choi and S.Y.Lee,“Secretory and extracellular production of recombinant proteins using Escherichia coli,”Appl Microbiol Biotechnol,vol.64,no.5,pp.625–635,Jun.2004.
[3]S.C.Makrides,“Strategies for achieving high-level expression of genes in Escherichia coli.,”Microbiol.Rev.,vol.60,no.3,pp.512–538,Sep.1996.
[4]F.J.M.
D.K.Summers,and G.A.Monteiro,“Recombinant protein secretion in Escherichia coli Biotechnology Advances,vol.23,no.3,pp.177–202,May 2005.
[5]Baneyx F.Recombinant protein expression in Escherichia coli.Current opinion in biotechnology.1999;10(5):411–421.
[6]Terpe K.Overview of bacterial expression systems for heterologous protein production:from molecular and biochemical fundamentals to commercial systems.Appl Microbiol Biotechnol.2006;72(2):211–222
[7]Contreras-Gomez A,Sanchez-Miron A,Garcia-Camacho F,Molina-Grima E,Chisti Y.Protein production using the baculovirus-insect cell expression system.Biotechnol Prog.2014;30(1):1–18.
[8]Dalton AC,Barton WA.Over-expression of secreted proteins from mammalian cell lines.Protein Sci.2014;23(5):517–525
[9]Legendre JY,Szoka FC.Cyclic amphipathic peptide-DNA complexes mediate high-efficiency transfection of adherent mammalian cells.Proc Natl Acad Sci U S A.1993;90(3):893–897.
[10]Kimple,M.E.,Brill,A.L.,&Pasker,R.L.(2013).Overview of affinity tags for protein purification.Current protocols in protein science,73,Unit–9.9.
[11]G.Garke,W.-D.Deckwer,and F.B.Anspach,“Preparative two-step purification of recombinant human basic fibroblast growth factor from high-cell-density cultivation of Escherichia coli,”Journal of Chromatography B:Biomedical Sciences and Applications,vol.737,no.1–2,pp.25–38,Jan.2000.
[12]J.A.Andrades,J.A.Santamaría,L.T.Wu,F.L.Hall,M.E.Nimni,and J.Becerra,“Production of a recombinant human basic fibroblast growth factor with a collagen binding domain,”Protoplasma,vol.218,no.1,pp.95–103,2001.
[13]D.B.Smith and K.S.Johnson,“Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase,”Gene,vol.67,no.1,pp.31–40,Jul.1988.
[14]K.Guan and J.E.Dixon,“Eukaryotic proteins expressed in Escherichia coli:Animproved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase,”Analytical Biochemistry,vol.192,no.2,pp.262–267,Feb.1991.
[15]Lin,V.C.,Huang,S.P.,Ting,H.J.,Ma,W.L.,Yu,C.C.,Huang,C.Y.,...&Lu,T.L.(2017).Vitamin D receptor-binding site variants affect prostate cancer progression.Oncotarget,8(43),74119.
[16]Ross-Innes,C.S.,Stark,R.,Teschendorff,A.E.,Holmes,K.A.,Ali,H.R.,Dunning,M.J.,…Carroll,J.S.(2012).Differential oestrogen receptor binding is associated with clinical outcome in breast cancer.Nature,481(7381),389–393.
[17]Samuel B.Lehrer,W.Elliott Horner,Gerald Reese&Dr.Steven Taylor(1996)Why are some proteins allergenic?Implications for biotechnology,Critical Reviews in Food Science and Nutrition,36:6,553-564
[18]Smoldovskaya,O.,Feyzkhanova,G.,Arefieva,A.,Voloshin,S.,Ivashkina,O.,Reznikov,Y.,&Rubina,A.(2016).Allergen extracts and recombinant proteins:comparison of efficiency of in vitro allergy diagnostics using multiplex assay on a biological microchip.Allergy,asthma,and clinical immunology:official journal of the Canadian Society of Allergy and Clinical Immunology,12,9.
[19]L.Wang,J.H.Kang,K.H.Kim,and E.K.Lee,“Expression of intein‐tagged fusion protein and its applications in downstream processing,”Journal of Chemical Technology and Biotechnology,vol.85,no.1,pp.11–18,Jan.2010.
[20]S.Elleuche and S.
“Inteins,valuable genetic elements in molecular biology and biotechnology,”Applied Microbiology and Biotechnology,vol.87,no.2,pp.479–489,Jun.2010.
[21]Y.Li,“Self-cleaving fusion tags for recombinant protein production,”Biotechnology Letters,vol.33,no.5,pp.869–881,May 2011.
[22]A.b.Ingham,K.w.Sproat,M.l.v.Tizard,and R.j.Moore,“Aversatile system for the expression of nonmodified bacteriocins in Escherichia coli,”Journal of Applied Microbiology,vol.98,no.3,pp.676–683,2005.
[23]I.R.Cottingham,A.Millar,E.Emslie,A.Colman,A.E.Schnieke,and C.McKee,“Amethod for the amidation of recombinant peptides expressed as intein fusion proteins in Escherichia coli,”Nature Biotechnology,vol.19,no.10,pp.974–977,Oct.2001.
[24]M.P.Zeidler,C.Tan,Y.Bellaiche,S.Cherry,S.
U.Gayko,and N.Perrimon,“Temperature-sensitive control of protein activity by conditionally splicing inteins,”Nat Biotech,vol.22,no.7,pp.871–876,Jul.2004.
[25]O.Király,L.Guan,E.Szepessy,M.Tóth,Z.Kukor,and M.Sahin-Tóth,“Expression of human cationic trypsinogen with an authentic N terminus using intein-mediated splicing in aminopeptidase P deficient Escherichia coli,”Protein Expression and Purification,vol.48,no.1,pp.104–111,Jul.2006.
Claims (12)
- 一种用于表达FGF-2的DNA序列,其特征在于,该序列为SEQ ID NO.1所示或者为其互补序列。
- 一种用于表达FGF-2的DNA序列,其特征在于,该序列为SEQ ID NO.2所示或者为其互补序列。
- 一种构建体,其特征在于,该构建体包括质粒载体EcoRI和NotI限制性酶切位点之间的如SEQ ID NO.1所示DNA序列或者为其互补序列,或者该构建体包括质粒载体EcoRI和NotI限制性酶切位点之间的如SEQ ID NO.2所示DNA序列或者为其互补序列。
- 根据权利要求3所述的构建体,其特征在于,所述质粒载体为pcDNA 3.1(+)或者pNC1。
- 根据权利要求3所述的构建体,其特征在于,所述构建体包括质粒载体EcoRI和NotI限制性酶切位点之间的如SEQ ID NO.2所示DNA序列或者为其互补序列,所述质粒载体为pNC1。
- 一种构建体的制备方法,其特征在于,所述方法包括以下步骤:a.合成含有EcoRI和NotI限制性酶切位点的如SEQ ID NO.1所示的DNA序列或其互补序列;或者合成含有EcoRI和NotI限制性酶切位点的如SEQ ID NO.2所示的DNA序列或其互补序列;b.用EcoRI和NotI消化质粒载体,回收大片段;c.采用连接酶连接步骤a获得的DNA序列或者其互补序列与步骤b获得的大片段。d.将步骤c获得的连接产物转染细胞,筛选阳性克隆,提取质粒。
- 根据权利要求6所述的构建体的制备方法,其特征在于,所述质粒载体为pcDNA 3.1(+)或者pNC1。
- 权利要求3或4所述的构建体在表达FGF-2中的用途。
- 一种制备FGF-2的方法,其特征在于,该方法包括:a.培养细胞:培养哺乳动物细胞;b.转染:对a步骤获得的哺乳动物细胞进行转染如权利要求3-5中任一项所述的构建体;c.纯化:通过亲和层析,纯化FGF-2。
- 根据权利要求9所述的制备FGF-2的方法,其特征在于,所述亲和层析通过肝素结合结构域进行。
- 根据权利要求9所述的制备FGF-2的方法,其特征在于,在所述步骤b中,对a步骤获得的哺乳动物细胞进行转染如权利要求5所述的构建体;所述亲和层析为通过内含肽DnaE介导的亲和层析。
- 根据权利要求9-11任一项所述的制备FGF-2的方法,其特征在于,所述哺乳动物细胞为HEK 293T。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910543546.4 | 2019-06-21 | ||
CN201910543546.4A CN110777148B (zh) | 2019-06-21 | 2019-06-21 | 用于表达fgf-2的dna序列和制备fgf-2的方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2020253287A1 true WO2020253287A1 (zh) | 2020-12-24 |
Family
ID=69383271
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2020/079897 WO2020253287A1 (zh) | 2019-06-21 | 2020-03-18 | 优化的fgf2基因功能区序列及其在制备人fgf2中的应用 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN110777148B (zh) |
WO (1) | WO2020253287A1 (zh) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110777148B (zh) * | 2019-06-21 | 2021-08-06 | 梦芊科技知识产权有限公司 | 用于表达fgf-2的dna序列和制备fgf-2的方法 |
CN110777165B (zh) * | 2019-06-21 | 2021-08-06 | 梦芊科技知识产权有限公司 | 增强子序列和质粒载体及其制备方法和应用与转化体 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002022779A2 (en) * | 2000-09-18 | 2002-03-21 | Prochon Biotech Ltd. | High level expression of heterologous proteins |
CN103865932A (zh) * | 2012-12-11 | 2014-06-18 | 武汉禾元生物科技有限公司 | 一种从水稻种子生产重组人碱性成纤维细胞生长因子的方法 |
CN105143451A (zh) * | 2013-04-03 | 2015-12-09 | 允强·R·黄 | 在大肠杆菌的细胞质和/或培养基中表达真人类表皮生长因子和/或碱性成纤维细胞生长因子的手段和方法 |
CN110777148A (zh) * | 2019-06-21 | 2020-02-11 | 梦芊科技知识产权有限公司 | 优化的fgf2基因功能区序列来制造人类胚胎细胞因子-1 |
-
2019
- 2019-06-21 CN CN201910543546.4A patent/CN110777148B/zh active Active
-
2020
- 2020-03-18 WO PCT/CN2020/079897 patent/WO2020253287A1/zh active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002022779A2 (en) * | 2000-09-18 | 2002-03-21 | Prochon Biotech Ltd. | High level expression of heterologous proteins |
CN103865932A (zh) * | 2012-12-11 | 2014-06-18 | 武汉禾元生物科技有限公司 | 一种从水稻种子生产重组人碱性成纤维细胞生长因子的方法 |
CN105143451A (zh) * | 2013-04-03 | 2015-12-09 | 允强·R·黄 | 在大肠杆菌的细胞质和/或培养基中表达真人类表皮生长因子和/或碱性成纤维细胞生长因子的手段和方法 |
CN110777148A (zh) * | 2019-06-21 | 2020-02-11 | 梦芊科技知识产权有限公司 | 优化的fgf2基因功能区序列来制造人类胚胎细胞因子-1 |
Non-Patent Citations (3)
Title |
---|
XIUHUA HU, CHEUK YIN NELSON LAI, T. SIVAKUMAR, HAO WANG, K. L. NG, C. C. LAM , W. K. R. WONG: "Novel strategy for expression of authentic and bioactive human basic fibroblast growth factor in Bacillus subtilis", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 102, no. 16, 27 June 2018 (2018-06-27), pages 7061 - 7069, XP036557451, ISSN: 0157-7598, DOI: 10.1007/s00253-018-9176-1 * |
YANG, CHANGWEI: "Construction of Eukaryotic Expression Vector of Human bFGF, KGF and Their Expression in 293T Cells", CHINESE DOCTORAL DISSERTATIONS & MASTER'S THESES FULL-TEXT DATABASE, no. 9, 15 September 2016 (2016-09-15), pages 1 - 57, XP055766529, ISSN: 1671-6779 * |
ZHANG JIAO;CUI WEN-JING;MA XIANG-MIN;WANG WEN-WEN;WANG XIN: "Construction of Npu DnaE intein and its highly efficient protein trans-splicing activity in 293T cells", SHANDONG MEDICAL JOURNAL, vol. 53, no. 44, 29 November 2013 (2013-11-29), pages 10 - 13+122, XP055766535, ISSN: 1002-266x * |
Also Published As
Publication number | Publication date |
---|---|
CN110777148A (zh) | 2020-02-11 |
CN110777148B (zh) | 2021-08-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Meier et al. | A nuclear localization signal binding protein in the nucleolus. | |
Harris et al. | Assessing genetic heterogeneity in production cell lines: detection by peptide mapping of a low level Tyr to Gln sequence variant in a recombinant antibody | |
JPS6028994A (ja) | 〔21―ロイシン〕ヒトウロガストロン | |
JPS62246600A (ja) | ヒト上皮成長因子およびその類似物質の産生法 | |
KR20150008852A (ko) | 계면활성제 펩타이드의 제조 방법 | |
WO2020253287A1 (zh) | 优化的fgf2基因功能区序列及其在制备人fgf2中的应用 | |
JP6748061B2 (ja) | 二重シストロン細菌発現システム | |
JPWO2003091429A1 (ja) | 抗菌性ポリペプチド及びその利用 | |
EP1220933B1 (en) | Purification of recombinant proteins fused to multiple epitopes | |
KR102138272B1 (ko) | BiP 단편을 포함하는 재조합 벡터 및 상기 벡터를 이용한 재조합 단백질의 제조 방법 | |
WO2020253400A1 (zh) | 一种通过增强子促进fgf-2在293t细胞中表达的方法 | |
WO2020094005A1 (zh) | 重组Martentoxin的制备、表征及应用 | |
JP6302415B2 (ja) | ヒト上皮細胞増殖因子の製造方法 | |
US20190352365A1 (en) | Expression construct and method for producing proteins of interest | |
Razis et al. | The periplasmic expression of recombinant human epidermal growth factor (hEGF) in Escherichia coli | |
KR102060881B1 (ko) | 재조합 인간 혈청 알부민의 수용성 과발현 및 정제 방법 | |
CN109053875B (zh) | 突变型igf-1、重组质粒、重组蛋白及应用 | |
Hanif et al. | Heterologous secretory expression and characterization of dimerized bone morphogenetic protein 2 in Bacillus subtilis | |
JP2561122B2 (ja) | 機能性ポリペプチド | |
US20160333386A1 (en) | Use of peptidylglycine alpha-amidating monooxigenase (pam) for c-terminal amidation | |
US9676816B2 (en) | Heparin affinity tag and applications thereof | |
Wawiórka et al. | In vivo formation of Plasmodium falciparum ribosomal stalk—a unique mode of assembly without stable heterodimeric intermediates | |
CN101775404A (zh) | 一种原核表达体系高表达碱性蛋白的方法 | |
CN101096383A (zh) | 表皮生长因子与绿色荧光蛋白的融合蛋白 | |
CN111808170A (zh) | 多肽、hla-dr蛋白及其制备方法和应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 20826029 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 20826029 Country of ref document: EP Kind code of ref document: A1 |