CN118388567A - Specific targeted tyrosinase, preparation method and application thereof - Google Patents
Specific targeted tyrosinase, preparation method and application thereof Download PDFInfo
- Publication number
- CN118388567A CN118388567A CN202410832098.0A CN202410832098A CN118388567A CN 118388567 A CN118388567 A CN 118388567A CN 202410832098 A CN202410832098 A CN 202410832098A CN 118388567 A CN118388567 A CN 118388567A
- Authority
- CN
- China
- Prior art keywords
- tyrosinase
- solid phase
- preparing
- preparation
- specific
- 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
- 102000003425 Tyrosinase Human genes 0.000 title claims abstract description 71
- 108060008724 Tyrosinase Proteins 0.000 title claims abstract description 71
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 230000008685 targeting Effects 0.000 claims abstract description 22
- 239000002537 cosmetic Substances 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims description 30
- 238000006243 chemical reaction Methods 0.000 claims description 27
- 238000005406 washing Methods 0.000 claims description 25
- 239000007790 solid phase Substances 0.000 claims description 19
- 230000015572 biosynthetic process Effects 0.000 claims description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- 108091034117 Oligonucleotide Proteins 0.000 claims description 15
- 238000003786 synthesis reaction Methods 0.000 claims description 15
- 238000005859 coupling reaction Methods 0.000 claims description 13
- 150000008300 phosphoramidites Chemical class 0.000 claims description 12
- 239000000178 monomer Substances 0.000 claims description 10
- 238000007254 oxidation reaction Methods 0.000 claims description 10
- 125000006239 protecting group Chemical group 0.000 claims description 9
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 8
- 239000011574 phosphorus Substances 0.000 claims description 8
- 229910052698 phosphorus Inorganic materials 0.000 claims description 8
- 150000003536 tetrazoles Chemical class 0.000 claims description 8
- 230000008878 coupling Effects 0.000 claims description 7
- 238000010168 coupling process Methods 0.000 claims description 7
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 7
- 238000010511 deprotection reaction Methods 0.000 claims description 6
- 230000003647 oxidation Effects 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 5
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims description 4
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 abstract description 62
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 abstract description 54
- 230000002087 whitening effect Effects 0.000 abstract description 17
- 108020004999 messenger RNA Proteins 0.000 abstract description 10
- 210000000170 cell membrane Anatomy 0.000 abstract description 7
- 239000002502 liposome Substances 0.000 abstract description 6
- 230000015556 catabolic process Effects 0.000 abstract description 5
- 238000006731 degradation reaction Methods 0.000 abstract description 5
- 239000007921 spray Substances 0.000 abstract description 5
- 238000007385 chemical modification Methods 0.000 abstract description 4
- 239000002674 ointment Substances 0.000 abstract description 3
- 239000008176 lyophilized powder Substances 0.000 abstract description 2
- 239000003981 vehicle Substances 0.000 abstract 1
- 238000001514 detection method Methods 0.000 description 38
- 150000007523 nucleic acids Chemical class 0.000 description 34
- 108020004707 nucleic acids Proteins 0.000 description 32
- 102000039446 nucleic acids Human genes 0.000 description 32
- 238000012360 testing method Methods 0.000 description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 31
- 230000000692 anti-sense effect Effects 0.000 description 27
- 102000004169 proteins and genes Human genes 0.000 description 27
- 108090000623 proteins and genes Proteins 0.000 description 27
- 239000000243 solution Substances 0.000 description 24
- 239000000523 sample Substances 0.000 description 23
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 19
- 239000013642 negative control Substances 0.000 description 19
- 238000010438 heat treatment Methods 0.000 description 18
- 239000007788 liquid Substances 0.000 description 18
- 238000012546 transfer Methods 0.000 description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 16
- 238000001962 electrophoresis Methods 0.000 description 16
- 239000001963 growth medium Substances 0.000 description 15
- 238000002156 mixing Methods 0.000 description 15
- 239000002609 medium Substances 0.000 description 14
- 230000005764 inhibitory process Effects 0.000 description 13
- 239000000499 gel Substances 0.000 description 12
- 239000013641 positive control Substances 0.000 description 12
- 235000019441 ethanol Nutrition 0.000 description 11
- 239000012634 fragment Substances 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 10
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 10
- 239000012153 distilled water Substances 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- 230000006872 improvement Effects 0.000 description 9
- 239000006285 cell suspension Substances 0.000 description 8
- 238000011534 incubation Methods 0.000 description 8
- 238000004113 cell culture Methods 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 210000003491 skin Anatomy 0.000 description 7
- 238000001890 transfection Methods 0.000 description 7
- 241000252212 Danio rerio Species 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000011068 loading method Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 230000004048 modification Effects 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- 239000010902 straw Substances 0.000 description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 6
- 238000010257 thawing Methods 0.000 description 6
- 102000004142 Trypsin Human genes 0.000 description 5
- 108090000631 Trypsin Proteins 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 230000027832 depurination Effects 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 238000007710 freezing Methods 0.000 description 5
- 230000008014 freezing Effects 0.000 description 5
- 230000001678 irradiating effect Effects 0.000 description 5
- BEJNERDRQOWKJM-UHFFFAOYSA-N kojic acid Chemical compound OCC1=CC(=O)C(O)=CO1 BEJNERDRQOWKJM-UHFFFAOYSA-N 0.000 description 5
- 229960004705 kojic acid Drugs 0.000 description 5
- WZNJWVWKTVETCG-UHFFFAOYSA-N kojic acid Natural products OC(=O)C(N)CN1C=CC(=O)C(O)=C1 WZNJWVWKTVETCG-UHFFFAOYSA-N 0.000 description 5
- 230000036564 melanin content Effects 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 229940121649 protein inhibitor Drugs 0.000 description 5
- 239000012268 protein inhibitor Substances 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 239000012588 trypsin Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 238000002123 RNA extraction Methods 0.000 description 4
- 239000013614 RNA sample Substances 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 125000004043 oxo group Chemical group O=* 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 125000004149 thio group Chemical group *S* 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 240000001624 Espostoa lanata Species 0.000 description 3
- 235000009161 Espostoa lanata Nutrition 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 238000000692 Student's t-test Methods 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 3
- 239000012578 cell culture reagent Substances 0.000 description 3
- 230000007673 developmental toxicity Effects 0.000 description 3
- 231100000415 developmental toxicity Toxicity 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 201000001441 melanoma Diseases 0.000 description 3
- 239000008057 potassium phosphate buffer Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000010079 rubber tapping Methods 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 238000004062 sedimentation Methods 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- 230000001502 supplementing effect Effects 0.000 description 3
- 238000012353 t test Methods 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- 108020004463 18S ribosomal RNA Proteins 0.000 description 2
- 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 2
- 125000004200 2-methoxyethyl group Chemical group [H]C([H])([H])OC([H])([H])C([H])([H])* 0.000 description 2
- 108020005075 5S Ribosomal RNA Proteins 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- 206010070834 Sensitisation Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000007984 Tris EDTA buffer Substances 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 150000002500 ions Chemical group 0.000 description 2
- 230000007794 irritation Effects 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 238000011880 melting curve analysis Methods 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 230000000149 penetrating effect Effects 0.000 description 2
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 230000008313 sensitization Effects 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 125000004434 sulfur atom Chemical group 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000001521 two-tailed test Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 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
- 125000001731 2-cyanoethyl group Chemical group [H]C([H])(*)C([H])([H])C#N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 101000611536 Agaricus bisporus Polyphenol oxidase 3 Proteins 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 102000030523 Catechol oxidase Human genes 0.000 description 1
- 108010031396 Catechol oxidase Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 1
- KDCGOANMDULRCW-UHFFFAOYSA-N Purine Natural products N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 1
- CVQUWLDCFXOXEN-UHFFFAOYSA-N Pyran-4-one Chemical compound O=C1C=COC=C1 CVQUWLDCFXOXEN-UHFFFAOYSA-N 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 101000611541 Sarcophaga argyrostoma Phenoloxidase 3 Proteins 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000001816 cooling 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
- 108010034748 copper-binding protein Proteins 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005485 electric heating Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- PSLIMVZEAPALCD-UHFFFAOYSA-N ethanol;ethoxyethane Chemical compound CCO.CCOCC PSLIMVZEAPALCD-UHFFFAOYSA-N 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 239000011539 homogenization buffer Substances 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000004249 ion pair reversed phase high performance liquid chromatography Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000008099 melanin synthesis Effects 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 238000002381 microspectrum Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 1
- DIHKMUNUGQVFES-UHFFFAOYSA-N n,n,n',n'-tetraethylethane-1,2-diamine Chemical compound CCN(CC)CCN(CC)CC DIHKMUNUGQVFES-UHFFFAOYSA-N 0.000 description 1
- -1 nickel sulfate amine Chemical class 0.000 description 1
- 229910000363 nickel(II) sulfate Inorganic materials 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 150000008301 phosphite esters Chemical class 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 230000019612 pigmentation Effects 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 210000000434 stratum corneum Anatomy 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 125000000464 thioxo group Chemical group S=* 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- AVBGNFCMKJOFIN-UHFFFAOYSA-N triethylammonium acetate Chemical compound CC(O)=O.CCN(CC)CC AVBGNFCMKJOFIN-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- KAKZBPTYRLMSJV-UHFFFAOYSA-N vinyl-ethylene Natural products C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
-
- 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
- A61K8/66—Enzymes
-
- 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
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/18—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with another compound as one donor, and incorporation of one atom of oxygen (1.14.18)
- C12Y114/18001—Tyrosinase (1.14.18.1)
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Birds (AREA)
- Dermatology (AREA)
- Epidemiology (AREA)
- Virology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a specific targeting tyrosinase, a preparation method and application thereof, wherein the specific targeting tyrosinase mRNA degrades hTYR mRNA sequences, and inhibits the expression of tyrosinase, so that the generation of melanin is inhibited for a long time, the whitening effect is achieved, the specificity is high, the safety is high, the special chemical modification is high, the stability is high, and the degradation is not easy; without liposome delivery vehicles, the liposome can automatically penetrate through cell membranes and enter the inside of the cells to play a role. The effective use concentration is low, the cost is controllable, and the use is convenient: can be used alone or in combination with other conventional cosmetics, and is suitable for various product forms such as ointment, spray or lyophilized powder.
Description
Technical Field
The invention relates to the fields of cosmetics, medicines and food chemistry, in particular to specific targeted tyrosinase, a preparation method and application thereof.
Background
Tyrosinase (EC 1.14.18.1, tyrosinase, abbreviated as TYR) is also known as polyphenol oxidase, and is a copper binding protein. The enzyme is a key enzyme protein causing pigmentation of human skin and related diseases.
The research of inhibitors for tyrosinase activity has been paid strong attention to a plurality of fields at home and abroad for many years, but in a plurality of existing compounds applied to inhibiting tyrosinase activity, practice proves that most of the compounds have the problems of low efficacy and obvious toxic and side effects.
Kojic acid (Kojic acid), chemical name 5-hydroxy-2-hydroxymethyl-1, 4-pyrone. The kojic acid has the function of inhibiting the tyrosinase activity of melanin-producing enzyme, has remarkable whitening effect, does not inhibit other enzymes, and has wide application value in the daily chemical industry field. Traditional raw materials of whitening cosmetics, such as kojic acid and the like, have certain whitening effects, but have the defects of safety problems, unstable effects and the like.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide specific targeted tyrosinase, a preparation method and application thereof.
The technical scheme adopted for solving the technical problems is as follows: the preparation method of the specific targeted tyrosinase comprises the following preparation and synthesis routes:
，
Wherein the synthesis step comprises deprotection, coupling, oxidation and capping reaction steps;
Wherein, Is a solid phase synthetic linkage.
As a further improvement of the invention: wherein the deprotection step is of the formula:
；
Synthesizing the 3' -end first base of the oligonucleotide, selecting CPG linked with one base in A, G, C, T as a solid phase carrier, protecting the 5' OH of the base by a DMT protecting group, removing DMT of the first base by trichloroacetic acid before starting synthesis, and exposing active 5' OH; wherein the method comprises the steps of Is a solid phase synthetic linkage.
As a further improvement of the invention: the coupling reaction formula is as follows:
；
The phosphite ester linkage formed in the coupling step is unstable under acidic conditions, and requires the oxidation of trivalent phosphorus to pentavalent phosphorus with I2 to form a stable phosphodiester linkage. There are two ways of oxidation: oxo and thioxo; and (3) cleaning residual trichloroacetic acid on the solid phase carrier by using anhydrous acetonitrile, adding tetrazole and corresponding phosphoramidite monomers, and reacting the phosphoramidite monomers activated by tetrazole with 5' OH on the solid phase carrier to form a phosphoramidite bond. To ensure that only one base is coupled per cycle, the phosphoramidite monomer 5' OH is protected by DMT; wherein the method comprises the steps of Is a solid phase synthetic linkage.
As a further improvement of the invention: the oxidation reaction formula is as follows:
Or (b)
；
I2 oxidizes trivalent phosphorus to pentavalent phosphorus, forming a phosphodiester bond; after DMT removal is completed, residual trichloroacetic acid on the solid phase carrier is cleaned by anhydrous acetonitrile, tetrazole and corresponding phosphoramidite monomers are added, and the phosphoramidite monomers activated by tetrazole react with 5' OH on the solid phase carrier to form a phosphoramidite bond.
As a further improvement of the invention: the capping reaction formula is as follows:
；
the 5'OH which does not participate in the reaction is blocked by acetyl, the coupling efficiency of the phosphoramidite method for synthesizing the oligonucleotide can reach more than 99.5 percent on average in each cycle, but the coupling efficiency can not reach 100 percent in the perfect synthesis process, and each cycle of coupling reaction has a trace of 5' OH which does not participate in the coupling reaction, and the 5'OH which does not participate in the reaction needs to be blocked by acetyl so as to avoid the participation of the 5' OH in the next cycle of reaction; wherein the method comprises the steps of Is a solid phase synthetic linkage.
As a further improvement of the invention: the deprotection, coupling, oxidation and capping steps are cycled, with each cycle reacting one base until the entire sequence is complete.
As a further improvement of the invention: the method also comprises the step of cutting off the solid phase carrier, wherein the reaction formula is as follows:
；
as a further improvement of the invention: also included is the cleavage amino protecting group chemical reaction equation:
；
The chemical reaction equation for removing the phosphate skeleton protecting group is as follows:
。
as a further improvement of the invention: chemical synthesis of oligonucleotides usually uses butadiene amino groups to immobilize the 3 'end of the oligonucleotide on a solid support, all reagents in the synthesis of the groups are stable, and after synthesis, the groups can be cleaved off with ammonia to separate the oligonucleotide from the solid support, forming an oligonucleotide with 3' OH. Meanwhile, ammonia water is used for removing the protecting groups of pyrimidine and purine primary amino groups and cyanoethyl protecting groups on the phosphate skeleton of the oligonucleotide at room temperature, and only the oligonucleotide with completely excised amino protecting groups and phosphate skeleton protecting groups has biological activity.
The invention also comprises the specific targeting tyrosinase, and the specific targeting tyrosinase is prepared by the preparation method.
The invention also comprises application of the specific targeting tyrosinase prepared by the preparation method of the specific targeting tyrosinase in preparation of cosmetics.
Compared with the prior art, the invention has the beneficial effects that:
1. The specific targeting tyrosinase provided by the invention can specifically target tyrosinase mRNA, and can inhibit the expression of tyrosinase, so that the production of melanin is inhibited for a long time, the whitening effect is achieved, the hTYR mRNA sequences are specifically degraded, the specificity is high, the safety is high, the special chemical modification is performed, the stability is high, and the degradation is difficult;
2. the liposome delivery carrier is not needed, and the liposome can automatically penetrate through cell membranes and enter the inside of the cells to play a role;
3. The antisense nucleic acid designs a plurality of antisense nucleic acid sequences in a tyrosinase mRNA single-chain region by combining with the latest RNA advanced structure prediction algorithm, and a sequence with high inhibition efficiency and strong specificity is obtained after screening;
4. The 3' -end 4 bases and the 5' -end 4 bases of the antisense nucleic acid are modified by adopting a Locked Nucleic Acid (LNA) or modified by 2' -O- (2-Methoxyethyl) so as to improve the complementary binding capacity of the antisense nucleic acid and the tyrosinase mRNA sequence and effectively enhance the stability and affinity of the antisense nucleic acid; the middle is 8-10 DNA bases, the whole chain is modified by phosphorothioate, the biological stability is improved, and the phosphorothioate modification greatly improves the stability of the antisense nucleic acid in organisms;
5. Since phosphorothioate linkages are more resistant to nuclease degradation than unmodified phosphate linkages (p=o), this allows the modified antisense nucleic acid to be present in vivo for a longer period of time, thereby enhancing its inhibitory effect. Phosphorothioate modifications may also enhance the binding affinity of antisense nucleic acids to target RNAs. This is because the introduction of sulfur atoms alters the steric structure of the antisense nucleic acid molecule, helping it to bind more stably to the target RNA.
Phosphorothioate modified antisense nucleic acids are more readily taken up by cells. Such modifications alter the negative charge distribution of antisense nucleic acids, helping them to pass through the cell membrane, thereby increasing intracellular concentration. The 3 'end or the 5' end of the antisense nucleic acid is chemically modified by cholesterol or C16, so that the cell membrane penetrating effect of the antisense nucleic acid can be improved, and the antisense nucleic acid can enter cells to play a role without the assistance of other delivery materials.
Drawings
For a clearer description of the technical solutions, the drawings that are required to be used in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a western blotting assay in a melanin inhibition assay for a TRY protein inhibitor specifically targeting tyrosinase according to the present invention.
FIG. 2 is a diagram showing a gray scale analysis of estern blotting detection in a melanin inhibition test of a TRY protein inhibitor specifically targeting tyrosinase according to the present invention.
FIG. 3 is a diagram of melanin detection analysis in a tyrosinase-specific TRY protein inhibitor melanin inhibition test of the present invention.
FIG. 4 shows the result of QRT-PCR detection in the result of a long-acting inhibition test of specific targeted tyrosinase according to the present invention.
FIG. 5 is a schematic representation of the expression level of tyrosinase in a long-acting inhibition test of a specific tyrosinase-targeted assay of the invention.
Fig. 6 is a schematic diagram showing the result of determining the L values of a model after the completion of the colorimetric detection in a specific tyrosinase-targeted whitening efficacy test, i.e., a melanin skin model whitening test.
Fig. 7 is a schematic diagram showing the summary of the color results of the specific tyrosinase targeting whitening efficacy test-melanin skin model whitening test.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments of the present invention and corresponding drawings. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention will now be further described with reference to the accompanying drawings and examples: the invention provides specific targeted tyrosinase, a preparation method and application thereof, which are obtained by a specific chemical synthesis method and are subjected to special chemical modification to improve the stability and the bioactivity of the specific targeted tyrosinase.
The preparation method of the specific targeted tyrosinase comprises the following preparation and synthesis routes:
，
The phosphoramidite method synthesizes from the 3 'to 5' end of the oligonucleotide, and one base is added in each cycle; each cycle reacts with one base until the entire sequence is completed.
The synthesis steps include deprotection, coupling, oxidation and capping reaction steps.
Wherein the deprotection reaction comprises the steps of selecting CPG linked with one base of A, G, C, T as a solid phase carrier according to the first base of the 3' end of the synthesized oligonucleotide, wherein the 5' OH of the base is protected by a DMT protecting group, and removing DMT of the first base by trichloroacetic acid before starting synthesis to expose active 5' OH, wherein the reaction equation is shown as follows:
。
The coupling reaction includes the steps of washing residual trichloroacetic acid on solid carrier with anhydrous acetonitrile after DMT elimination, adding tetrazole and corresponding phosphoramidite monomer, and reaction between tetrazole activated phosphoramidite monomer and 5' OH on solid carrier to form phosphoramidite bond. To ensure that only one base is coupled per cycle, the phosphoramidite monomer 5' OH is protected by DMT. Wherein the method comprises the steps of Is a solid phase synthetic linkage; the reaction equation is shown as follows:
。
Wherein, the oxidation reaction comprises the following steps that the oxidation reaction comprises oxo and thio, the phosphite ester bond formed after the coupling reaction is unstable under the acidic condition, and I2 is needed to oxidize trivalent phosphorus into pentavalent phosphorus to form stable phosphodiester bond; wherein the method comprises the steps of Is a solid phase synthetic linkage.
The oxo reaction equation is shown as follows:
；
the thio reaction equation is shown as follows:
。
as one embodiment of the invention, the method further comprises a purification step of purifying the oligonucleotide by adopting an ion-pair reversed-phase high performance liquid chromatography, wherein the characteristic of the difference of the hydrophobicity of the oligos with different lengths is utilized, and along with the enhancement of elution strength, the salt, the short fragment, the long fragment, the target fragment and the fragment with failed protecting group removal of the synthesis failure are sequentially eluted, so that the purpose of purifying the oligonucleotide is achieved, and the purity after purification can reach more than 95%.
Wherein the purification step is followed by vacuum drying.
As an embodiment of the invention, the specific targeting tyrosinase provided by the invention can be added into products with different forms such as ointment, spray or freeze-dried powder according to the need in a cosmetic formula. Specific methods of use include, but are not limited to: as effective components of whitening cosmetics, the composition is used for preparing whitening cream, whitening essence and the like; the sun-proof spray is prepared by combining sun-proof components, so that the whitening and sun-proof effects are provided.
According to the specific targeting tyrosinase provided by the invention, a plurality of antisense nucleic acid sequences are designed in a tyrosinase mRNA single-chain region, and a sequence with high inhibition efficiency and strong specificity is obtained after screening. The 3' -end 4 bases and the 5' -end 4 bases of the antisense nucleic acid are modified by adopting a Locked Nucleic Acid (LNA) or modified by 2' -O- (2-Methoxyethyl) so as to improve the complementary binding capacity of the antisense nucleic acid and a tyrosinase mRNA sequence and effectively enhance the stability and affinity of the antisense nucleic acid; the middle is 8-10 DNA bases, the whole chain is modified by phosphorothioate, the biostability is improved, and the phosphorothioate modification greatly improves the stability of the antisense nucleic acid in the organism. Since phosphorothioate linkages are more resistant to nuclease degradation than unmodified phosphate linkages (p=o), this allows the modified antisense nucleic acid to be present in vivo for a longer period of time, thereby enhancing its inhibitory effect. Phosphorothioate modifications may also enhance the binding affinity of antisense nucleic acids to target RNAs. This is because the introduction of sulfur atoms alters the steric structure of the antisense nucleic acid molecule, helping it to bind more stably to the target RNA. Phosphorothioate modified antisense nucleic acids are more readily taken up by cells. Such modifications alter the negative charge distribution of antisense nucleic acids, helping them to pass through the cell membrane, thereby increasing intracellular concentration. The 3 'end or the 5' end of the antisense nucleic acid is chemically modified by cholesterol or C16, so that the cell membrane penetrating effect of the antisense nucleic acid can be improved, and the antisense nucleic acid can enter cells to play a role without the assistance of other delivery materials.
The effective sequence of the specific targeted tyrosinase is tested, and the testing steps are as follows:
experimental materials: human melanoma cells SK-MEL-28 (supplied by Guangzhou light Biotechnology Co., ltd.);
tyrosinase ASO is provided by apparent biosynthesis.
Experimental reagent:
Cell culture reagent: fetal bovine serum (Hyclone, cat.no. sh30087.01), DMEM-high sugar medium (Hyclone, cat.no. sh30022.01 b), green streptomycin (Hyclone, cat.no. SH 30010), PBS potassium phosphate buffer (Hyclone, cat.no. sh30256.01 b).
Experiment consumable:
6-well plate cell culture plate (CORNING, cat.No.040810004), pasteur pipette (JET BIOFIL ml).
The testing method comprises the following steps:
Cell resuscitation (one)
1) Preheating the water bath to 37 ℃;
2) Wiping the surface of the ultra-clean workbench for 30min by using 75% alcohol;
3) Placing sterilized centrifuge tube, straw, culture flask, etc. in order in the ultra-clean workbench;
4) Taking out the frozen tube;
5) Rapidly thawing, namely rapidly putting the frozen storage tube into a preheated water bath for rapid thawing, continuously shaking to rapidly thaw the liquid in the tube, and taking out the tube when the frozen storage tube is slightly unmelted;
6) Wiping the outer wall of the freezing and storing tube with alcohol cotton balls, and then taking into an ultra-clean bench;
7) Preparing cell suspension, transferring cells into a 15ml centrifuge tube, adding preheated culture medium drop by drop, and shaking the centrifuge tube at the same time; the amount of the added culture medium is more than 10 ml;
8) Centrifuging at 800rpm for 5min on a low speed centrifuge; the supernatant was aspirated and the cells were resuspended in 1ml of medium;
9) Packaging the cell suspension into a culture dish, placing the culture dish into a culture box containing CO2 at 37 ℃ for culture, and changing the liquid for a time which depends on the sedimentation rate of the cells.
(II) cell culture
2.1 Preheating the water bath kettle to 37 ℃;
2.2 Wiping the surface of the ultra-clean workbench with 75% alcohol and irradiating for 30 min;
2.3 Placing sterilized centrifuge tubes, straws, culture bottles and the like in an ultra-clean workbench in sequence;
2.4 Taking out the cell culture flask, and performing aseptic operation;
2.5 Opening the bottle cap to suck the old culture solution;
2.6 Washing the cells with PBS one to two times;
2.7 For cell trypsinization): trypsin-EDTA solution (1 ml/25cm2,2ml/75cm 2) was added to the rinsed cells and the bottom of the cell dish was gently washed. Sucking the trypsin-EDTA solution, placing in a 37 ℃ incubator for 2-3 minutes, tapping the wall of the incubator to make most of the cells fall off, observing under an inverted microscope, and adding a proper amount of fresh culture medium containing serum to stop the action of the trypsin when the cells are to be separated and are in a round particle shape;
2.8 Sucking up and down for several times by a suction tube to break up cell clusters, uniformly mixing, supplementing 3n (n is the number of passage bottles) ml culture medium (MEM), and transferring to a new culture bottle according to a dilution ratio;
2.9 Placing into a CO2 incubator (culture conditions of 5% CO2, saturated humidity, 37 ℃), and replacing the culture medium every 3 days.
(III) screening ASO by cell transfection
3.1 ASO screening transfection protocol:
(1) And (3) paving: the density is 5 multiplied by 10 and the seed cells are planted in 5/hole, and the seed cells are fully and evenly shaken;
Sample setting:
Plate 1:
| NC ASO 25nM | NC ASO 50nM | NC ASO 100nM |
| Tyrosinase_001 ASO 25nM | Tyrosinase_001 ASO 50nM | Tyrosinase_001 ASO 100nM |
plate 2:
| Tyrosinase_002 ASO 25nM | Tyrosinase_002 ASO 50nM | Tyrosinase_002 ASO 100nM |
| Tyrosinase_003 ASO 25nM | Tyrosinase_003ASO 50nM | Tyrosinase_003 ASO 100nM |
(2) The third day the cell fusion degree is 40%, and transfection is carried out;
Transfection procedure:
a. cell exchange liquid: the complete medium was aspirated, washed twice with PBS, and 1ml of 20% foetal calf serum in DMEM high-sugar medium was added to each well;
b. Dissolving ASO with RNAase-free deionized water to a final concentration of 20uM, dissolving ASO in 500ul opti-MEM, mixing, and standing to obtain a tube-A;
| Final concentration | 25nM | 50nM | 100nM |
| Taking a quantity of 20uMASO | 2.5ul | 5.0ul | 10.0ul |
C. adding 5ul Lipofectamine ™ RNAiMAX into 500ul opti-MEM, gently mixing, standing for not more than 5 min-B tube;
d. mixing the tube A and the tube B, uniformly mixing, and standing for 20min;
e. respectively adding the mixture into each hole;
f. mixing, and placing into a cell incubator;
g. after 4-6 hours, the transfection medium was aspirated, washed twice with PBS, and 2ml of complete medium was added to each well;
(3) 24 hours after transfection, the medium was discarded, 1ml Trizol was added to each well, and quantitative PCR was prepared to detect the interference efficiency of ASO;
Note that (1) RNAase-free consumables must be used during lysis of ASO and ASO transfection.
(IV) PCR detection
Total RNA extraction
Collecting cells, adding 1ml of Trizol solution, blowing and mixing to make the cells fully lyse, and standing for 5min;
adding 200 μl of chloroform, shaking vigorously, mixing for 30s, allowing the water phase and the organic phase to fully contact, and standing at room temperature for 2min;
centrifuging at 4deg.C for 15min at 14,000g to obtain three layers, and transferring RNA into another new RNASE FREE EP tube in the upper water phase;
Precipitating RNA: adding isopropyl alcohol with the same volume, gently and fully mixing, and standing at room temperature for 10min;
centrifuging at 4deg.C for 10min at 14,000g, collecting RNA precipitate, and removing supernatant;
Washing twice with 75% ethanol, and air drying in an ultra clean bench;
The precipitate was dissolved by adding 20. Mu.l of DEPC water.
Second, total RNA purity and integrity detection
1) And (3) purity detection: taking 1 mul of RNA sample for 50-time dilution, and measuring an OD value on BioPhotometer plus Ai Bende nucleic acid protein tester, wherein the ratio of OD260/OD280 is more than 1.8, which indicates that the prepared RNA is purer and has no protein pollution;
2) Total RNA integrity detection: taking 1 μl of RNA sample, performing 1% agarose gel electrophoresis for 80V×20min, observing 5s rRNA, 18s rRNA and 28s rRNA bands of total RNA by using a gel imaging system, and if three bands are complete, proving that the total RNA extraction is complete.
Three, reverse transcription
The following solutions were prepared in RNASE FREE PCR tubes:
（EasyScript First-Strand cDNA Synthesis SuperMix）
(1) Incubating the 20 μl of the reaction solution at 25deg.C for 10min;
(2) Preserving the temperature at 42 ℃ for 30min;
(3) Preserving heat at 85 ℃ for 5s.
Fourth, quantitative PCR
1. Detecting sequence fragment size
Internal reference fragment: 18s-112bp;
the target fragment: tyrosinase-140bp;
| tyrosinase -F：5’ GGAACAAGCGAGTCGGATCT |
| tyrosinase -R：5’ GGAGTGGCTGCTTTTCTTCA |
| 18s-F：5’ CCTGGATACCGCAGCTAGGA |
| 18s-R：5’ GCGGCGCAATACGAATGCCCC |
2. the reaction system:
3. Reaction conditions:
95 ℃ for 5min; reading the plate at 95 ℃ for 15s and 60 ℃ for 32s, and performing 40 cycles;
Melting curve analysis: the temperature is 60-95 ℃;
Each sample was repeated 3 times;
Wherein, quantitative PCR appearance: ABI PRISM [ 7500 Sequence Detection System ]; suzhou Antai clean bench (SW-CJ-IFD), low speed centrifuge (Zhongjia, SC 3614), inverted light microscope (OLYMPUS CKX41, U-CTR 30-2), constant temperature incubator (Thermo scientific, HERACELL150 i) inverted fluorescence microscope manufacturer: leica model: DMI6000B.
The melanin inhibition test of the TRY protein inhibitor of the specific targeted tyrosinase comprises the following steps:
experimental materials: human melanoma cells SK-MEL-28 (supplied by Guangzhou light Biotechnology Co., ltd.);
tyrosinase ASO is provided by apparent biosynthesis.
Experimental reagent:
Cell culture reagent: fetal bovine serum (Hyclone, cat.no. sh30087.01), DMEM-high sugar medium (Hyclone, cat.no. sh30022.01 b), green streptomycin (Hyclone, cat.no. SH 30010), PBS potassium phosphate buffer (Hyclone, cat.no. sh30256.01 b).
The testing method comprises the following steps:
1. cell resuscitation
1.1. Preheating the water bath to 37 ℃;
1.2. Wiping the surface of the ultra-clean workbench with 75% alcohol and irradiating the ultra-clean workbench for 30min;
1.3. Placing sterilized centrifuge tubes, straws, culture bottles and the like in an ultra-clean workbench in sequence;
1.4. Taking out the freezing tube;
1.5. quickly thawing, namely quickly putting the frozen tube into a preheated water bath for quick thawing, continuously shaking to quickly thaw the liquid in the tube, and taking out the tube when the frozen tube is slightly unmelted;
1.6. Wiping the outer wall of the freezing and storing tube with alcohol cotton balls, and then taking into an ultra-clean bench;
1.7. preparing cell suspension, transferring cells into a 15ml centrifuge tube, adding preheated culture medium drop by drop, and shaking the centrifuge tube at the same time; the amount of the added culture medium is more than 10 ml;
1.8. centrifuging, centrifuging at 800rpm for 5 minutes on a low-speed centrifuge; the supernatant was aspirated and the cells were resuspended in 1ml of medium;
1.9. the cell suspension is split into culture dishes, the culture dishes are placed into a culture box containing CO2 at 37 ℃ for culture, and the liquid changing time is determined by the cell sedimentation speed.
2 Cell culture
2.1. Preheating the water bath to 37 ℃;
2.2. wiping the surface of the ultra-clean workbench with 75% alcohol and irradiating the ultra-clean workbench for 30min;
2.3. Placing sterilized centrifuge tubes, straws, culture bottles and the like in an ultra-clean workbench in sequence;
2.4. Taking out the cell culture bottle, and performing aseptic operation;
2.5. Opening the bottle cap to suck the old culture solution;
2.6. washing the cells with PBS one to two times;
2.7. For cell trypsinization: trypsin-EDTA solution (1 ml/25cm2,2ml/75cm 2) was added to the rinsed cells and the bottom of the cell dish was gently washed. Sucking the trypsin-EDTA solution, placing in a 37 ℃ incubator for 2-3 minutes, tapping the wall of the incubator to make most of the cells fall off, observing under an inverted microscope, and adding a proper amount of fresh culture medium containing serum to stop the action of the trypsin when the cells are to be separated and are in a round particle shape;
2.8. Sucking up and down for several times by a suction pipe to break up cell clusters, uniformly mixing, supplementing 3n (n is the number of passage bottles) ml culture medium (MEM), and transferring to a new culture bottle according to a dilution ratio;
2.9. The culture medium was replaced every 3 days in a CO2 incubator (culture conditions: 5% CO2, saturated humidity, 37 ℃).
3. Experimental grouping
| Cells | Experimental grouping | Detecting items |
| SK-MEL-28 | 1.200nM NC2.200nM ASO | 1. Transcriptome sequencing x repeat 32.Wb detection tyrosinase3. Biochemical detection of melanin |
。
4. Western blotting detection
4.1 Experimental materials
Antibody: anti-Tyrosinase antibody [ EPR10141] (ab 170905).
4.2 Experimental instrument
| Goods number/model | Name of the name | Manufacturer' s |
| mini protean 4 | BIO-RAD small-sized vertical electrophoresis tank | BIO-RAD (U.S.) |
| SW-CJ-1FD | Clean bench | Suzhou Antai air technologies Co., ltd (Suzhou) |
| TGL-16R | Low-temperature high-speed centrifugal machine | Pearl sea black horse (Pearl sea) |
| DK-8D type | Electric heating constant temperature water tank | Shanghai-Heng technology Co., ltd (Shanghai) |
| EPS100 | Electrophoresis apparatus | Shanghai Tencent technologies Co., ltd (Shanghai) |
| Tanon 1220 | Gel image analyzer | Shanghai Tencent technologies Co., ltd (Shanghai) |
| BioPhotometer plus | Nucleic acid protein tester | Eppendorf (Germany) |
| DHG-9140 | Electrothermal blowing drying box | Shanghai-Heng technology Co., ltd (Shanghai) |
| BL-50A | Vertical pressure sterilizer | Shanghai Boxun Utility Co., ltd (Shanghai) |
| 991 | Ultralow temperature refrigerator | Thermo (USA) |
。
4.3 Experimental method
4.3.1. Protein sample collection (Protein sample preparation)
Lysing adherent cells, suspension cells or tissue samples using an appropriate lysate RIPA lysate or the like;
after collection of the protein samples, it is necessary to determine the protein concentration of each protein sample in order to ensure consistent loading of each protein sample. Depending on the lysate used, appropriate protein concentration determination methods are required.
4.3.2. Electrophoresis (Electrophoresis)
(1) SDS-PAGE gel preparation
(2) Sample processing
An appropriate amount of concentrated SDS-PAGE protein loading buffer was added to the collected protein samples.
Heating at 100deg.C or boiling water bath for 3-5 min to thoroughly denature protein.
(3) Loading and electrophoresis
And (3) cooling to room temperature, and directly loading the protein sample into an SDS-PAGE gel loading hole.
In order to facilitate observation of the electrophoresis effect and the transfer effect, and judgment of the molecular weight of the protein, a pre-dye protein molecular weight standard is preferably used.
Electrophoresis is usually recommended to use low voltage constant voltage electrophoresis when the upper layer gel and high voltage constant voltage electrophoresis when bromophenol blue enters the lower layer gel. For a standard electrophoresis device of Bio-Rad or similar electrophoresis device, the low voltage may be set at 80-100V and the high voltage may be set at around 120V. For the convenience of electrophoresis, a constant voltage mode of the whole SDS-PAGE process can be adopted, the voltage is usually set at 100V, and then the timing time is set to be 90-120 minutes. Setting the timing can avoid frequent electrophoresis overhead.
In general, bromophenol blue reaches the vicinity of the bottom end of the gel to stop electrophoresis, or electrophoresis can be stopped after the target protein has been properly separated according to the electrophoresis condition of the pre-stained protein molecular weight standard.
4.3.3. Transfer film (Transfer)
Typically, if a standard wet film transfer apparatus of Bio-Rad is used, the film transfer current can be set to 300-400mA and the film transfer time can be set to 30-60 minutes. It can also be transferred overnight at 15-20 mA. The specific transfer time is determined according to the size of the target protein, and the longer the molecular weight of the target protein is, the smaller the molecular weight of the target protein is, and the shorter the transfer time is.
In the film transfer process, particularly when high-current rapid film transfer is performed, a very serious heating phenomenon usually occurs, and the film transfer groove is preferably placed in an ice bath for film transfer.
The effect of the transfer can be observed on the molecular weight standard of the pre-stained protein used, and usually the 1-2 bands with the largest molecular weight are difficult to transfer to the membrane entirely.
4.3.4. Closure (Blocking)
After the transfer of the membrane, the protein membrane is immediately placed into a prepared Western washing liquid, and rinsed for 1-2 minutes to wash the membrane transfer liquid. From all steps after finishing film transfer, care must be taken to keep the film moist, avoiding drying of the film, otherwise, a higher background is very easy to generate.
Slowly shake on a shaker and block at room temperature for 60 minutes. For some higher background antibodies, blocking was possible overnight at 4 ℃.
4.3.5. Anti-incubation (Primary antibody incubation)
Reference is made to the instructions for primary antibodies, in appropriate proportions, with Western primary antibody dilutions.
The blocking solution is sucked up by a miniature bench vacuum pump or a dropper, diluted primary antibody is immediately added, and the mixture is slowly shaken on a side swing table at room temperature or 4 ℃ for incubation for one hour. If the primary antibody is not effective for one hour, it may be incubated overnight with slow shaking at 4 ℃. Or further selecting an appropriate incubation temperature and time according to the instructions of the antibody.
Recovering the primary antibody. Western washing liquid is added, and the mixture is slowly shaken on a side swinging table for washing for 5 to 10 minutes. After the washing liquid is sucked out, the washing liquid is added for washing for 5 to 10 minutes. The washing was performed 3 times. If the result background is higher, the washing time can be prolonged and the washing times can be increased appropriately.
4.3.6. Second antibody incubation (Secondary antibody inucubation)
Referring to the instructions for the secondary antibodies, horseradish peroxidase (HRP) -labeled secondary antibodies were diluted with a Western secondary antibody diluent in appropriate proportions. The secondary antibody is selected according to the primary antibody, the washing liquid is sucked by a miniature desk vacuum pump or a dropper, and the diluted secondary antibody is immediately added, and the secondary antibody is slowly shaken on a side swinging table at room temperature or 4 ℃ for incubation for one hour.
Recovering the secondary antibody. Western washing liquid is added, and the mixture is slowly shaken on a side swinging table for washing for 5 to 10 minutes. After the washing liquid is sucked out, the washing liquid is added for washing for 5 to 10 minutes. The washing was performed 3 times. If the result background is higher, the washing time can be prolonged and the washing times can be increased appropriately.
4.3.7. Protein detection (Detection of proteins)
The proteins were detected using ECL-like reagents such as BeyoECL Plus. Tabletting may be performed using a dedicated tabletting cassette.
An X-ray film automatic film developing machine can be used for developing films. If the automatic film developing machine is not provided, the developing and fixing kit can be used for self-preparing developing solution and fixing solution for manual film developing. The X-ray film recommends the kodak X-OMAT BT film special for the biological experiment of the kodak original package.
4.3.8. Gel image analysis
The film is scanned or photographed and the molecular weight and net optical density values of the target band, such as the quality One of bio-rad, are analyzed using a gel image processing system.
5. Gray analysis of WB strips with ImagJ
6. Melanin content determination
6.1 With reference to the method proposed by Hideya Ando, etc., the melanin content is measured after a certain improvement. Adding PBS buffer solution into the standby cell suspension to adjust the cell density to 5 xl 0-5/ml;
6.2 Placing each group of 1 mL cell suspensions into 3 parallel colorimetric tubes with plugs, centrifuging, discarding supernatant, and adding 200 uL distilled water to resuspend the cells;
6.3 Then 1 mL 1:1 ethanol diethyl ether to dissolve non-melanin opaque particles, standing at room temperature for 15 min, centrifuging and discarding supernatant;
6.4 Adding 1mL NaOH solution with mass fraction of 2mol/L, and placing in water bath at 8O ℃ for 30min to dissolve cells;
6.5 The absorbance value was measured at 420 nm and divided by the number of cells as an index for evaluating the amount of melanin.
Melanin synthesis inhibition = [1- (drug treatment group value/cell number)/(blank group value/cell number) ] × 100%.
7. Experimental results
The result of the western blotting detection is shown in FIG. 1, and the grayscale analysis of the western blotting detection is shown in the following table:
| IOD | sample | 1 | 2 | 3 | mean±s.d. | t-test |
| Tyrosinase | NC Aso | 1745.81 | 1701.28 | 1769.51 | 1738.87±34.64 | |
| TYR Aso | 452.68 | 569.88 | 555.56 | 526.04±63.93 | ||
| GAPDH | NC Aso | 2179.38 | 2017.29 | 2199.30 | 2131.99±99.83 | |
| TYR Aso | 2112.62 | 2149.74 | 2094.50 | 2118.95±28.16 | ||
| Tyrosinase/GAPDH | NC Aso | 0.819 | 0.798 | 0.830 | 0.82±0.02 | |
| TYR Aso | 0.214 | 0.269 | 0.262 | 0.25±0.03 | 0.00 |
melanin detection and data analysis are shown in the following table:
| sample | 1 | 2 | 3 | 4 | 5 | 6 | mean±s.d. | Inhibition efficiency |
| NC-200 | 0.226 | 0.251 | 0.248 | 0.273 | 0.242 | 0.236 | 0.25±0.02 | 0.00% |
| si3-200 | 0.141 | 0.138 | 0.14 | 0.136 | 0.143 | 0.145 | 0.14±0 | 42.89% |
Wherein, the instrument adopts: suzhou Antai clean bench (SW-CJ-IFD), low speed centrifuge (Zhongjia, SC 3614), inverted light microscope (OLYMPUS CKX41, U-CTR 30-2), constant temperature incubator (Thermo scientific, HERACELL150 i) inverted fluorescence microscope manufacturer: leica model: DMI6000B.
The reagent is used:
1. SDS-PAGE reagent:
1.1 5 Xsample buffer (10 ml): 0.6ml 1mol/L Tris-HCl (pH 6.8), 5ml 50% glycerol, 2ml 10% SDS,0.5ml mercaptoethanol, 1ml 1% bromophenol blue, 0.9ml distilled water. Can be stored at 4deg.C for several weeks or at-20deg.C for several months;
1.2 Gel stock: in a fume hood, 30g of acrylamide, 0.8g of methylene bisacrylamide and 100ml of distilled water are weighed and dissolved. Filtering, placing in brown bottle, preserving at 4deg.C, and standing for 1 month;
1.3 Separation gel buffer at pH 8.9: tris 36.3g, adding 1mol/L HCl 48ml, adding 80ml distilled water to dissolve, adjusting pH to 8.9, fixing volume to 100ml, and preserving at 4 ℃;
1.4 Concentrated gum buffer at ph 6.7: tris 5.98g, adding 1mol/L HCl 48ml, adding 80ml distilled water to dissolve, adjusting pH to 6.7, fixing volume to 100ml, and preserving at 4 ℃;
1.5 TEMED (tetraethyl ethylenediamine) stock solution;
1.6 10% ammonium persulfate (freshly prepared with re-distilled water);
1.7 Tris-glycine electrode buffer pH 8.3: 6.0g of Tris, 28.8g of glycine and about 900ml of distilled water are weighed, the pH value is adjusted to 8.3, and the distilled water is used for fixing the volume to 1000ml. Stored at 4℃and diluted 10-fold before use.
2. Homogenization buffer: 1.0M Tris-HCl (pH 6.8) 1.Oml;10% SDS 6.0ml; beta-mercaptoethanol 0.2ml; ddH2O 2.8ml.
3. Transfer buffer: glycine 2.9g; tris 5.8g; SDS 0.37g; 200ml of methanol; ddH2O was added to volume 1000ml.
4.0.01M PBS (pH 7.4): 8.0g of NaCl; KCl 0.2g; 1.44g of Na2HPO 4; KH2PO4 0.24g; ddH2O was added to 1000ml.
5. Membrane staining solution: 0.2g of Coomassie Brilliant blue; 80ml of methanol; acetic acid 2ml; ddH2O118 ml. Coating liquid (5% skimmed milk powder, as prepared): 1.0g of skimmed milk powder was dissolved in 20ml of 0.01M PBS.
6. Color development liquid: DAB 6.0mg; 10.0ml of 0.01M PBS; nickel sulfate amine 0.1ml; h202 1.0 μl.
The melanin inhibition test of the TRY protein inhibitor of the specific targeted tyrosinase comprises the following steps:
experimental materials: human melanoma cells SK-MEL-28 (supplied by Guangzhou light Biotechnology Co., ltd.);
tyrosinase ASO is provided by apparent biosynthesis.
Experimental reagent:
Cell culture reagent: fetal bovine serum (Hyclone, cat.no. sh30087.01), DMEM-high sugar medium (Hyclone, cat.no. sh30022.01 b), green streptomycin (Hyclone, cat.no. SH 30010), PBS potassium phosphate buffer (Hyclone, cat.no. sh30256.01 b).
The testing method comprises the following steps:
1. Cell resuscitation
1.1. Preheating the water bath to 37 ℃;
1.2. Wiping the surface of the ultra-clean workbench with 75% alcohol and irradiating the ultra-clean workbench for 30min;
1.3. placing sterilized centrifuge tubes, straws, culture bottles and the like in an ultra-clean workbench in sequence;
1.4. Taking out the freezing tube;
1.5. quickly thawing, namely quickly putting the frozen tube into a preheated water bath for quick thawing, continuously shaking to quickly thaw the liquid in the tube, and taking out the tube when the frozen tube is slightly unmelted;
1.6. Wiping the outer wall of the freezing and storing tube with alcohol cotton balls, and then taking into an ultra-clean bench;
1.7. preparing cell suspension, transferring cells into a 15ml centrifuge tube, adding preheated culture medium drop by drop, and shaking the centrifuge tube at the same time; the amount of the added culture medium is more than 10 ml;
1.8. centrifuging, centrifuging at 800rpm for 5 minutes on a low-speed centrifuge; the supernatant was aspirated and the cells were resuspended in 1ml of medium;
1.9. the cell suspension is split into culture dishes, the culture dishes are placed into a culture box containing CO2 at 37 ℃ for culture, and the liquid changing time is determined by the cell sedimentation speed.
2. Cell culture
2.1. Preheating the water bath to 37 ℃;
2.2. wiping the surface of the ultra-clean workbench with 75% alcohol and irradiating the ultra-clean workbench for 30min;
2.3. Placing sterilized centrifuge tubes, straws, culture bottles and the like in an ultra-clean workbench in sequence;
2.4. Taking out the cell culture bottle, and performing aseptic operation;
2.5. Opening the bottle cap to suck the old culture solution;
2.6. washing the cells with PBS one to two times;
2.7. For cell trypsinization: trypsin-EDTA solution (1 ml/25cm2,2ml/75cm 2) was added to the rinsed cells and the bottom of the cell dish was gently washed. Sucking the trypsin-EDTA solution, placing in a 37 ℃ incubator for 2-3 minutes, tapping the wall of the incubator to make most of the cells fall off, observing under an inverted microscope, and adding a proper amount of fresh culture medium containing serum to stop the action of the trypsin when the cells are to be separated and are in a round particle shape;
2.8. Sucking up and down for several times by a suction pipe to break up cell clusters, uniformly mixing, supplementing 3n (n is the number of passage bottles) ml culture medium (MEM), and transferring to a new culture bottle according to a dilution ratio;
2.9 Placing into a CO2 incubator (culture conditions of 5% CO2, saturated humidity, 37 ℃) and replacing the culture medium every 3 days.
3 Experiment group
| Cells | Experimental grouping | Detecting items |
| SK-MEL-28 | 200NM ASO3 treated cells for 0, 4, 8, 12, 16 days | PCR detection tyrosinase |
。
4 PCR detection
Total RNA extraction
(1) Collecting cells, adding 1ml of Trizol solution, blowing and mixing to make the cells fully lyse, and standing for 5min;
(2) Adding 200 μl of chloroform, shaking vigorously, mixing for 30s, allowing the water phase and the organic phase to fully contact, and standing at room temperature for 2min;
(3) Centrifuging at 4deg.C for 15min at 14,000g to obtain three layers, and transferring RNA into another new RNASE FREE EP tube in the upper water phase;
(4) Precipitating RNA: adding isopropyl alcohol with the same volume, gently and fully mixing, and standing at room temperature for 10min;
(5) Centrifuging at 4deg.C for 10min at 14,000g, collecting RNA precipitate, and removing supernatant;
(6) Washing twice with 75% ethanol, and air drying in an ultra clean bench;
(7) The precipitate was dissolved by adding 20. Mu.l of DEPC water.
Second, total RNA purity and integrity detection
1) And (3) purity detection: taking 1 mul of RNA sample for 50-time dilution, and measuring OD value on BioPhotometer plus Ai Bende nucleic acid protein tester, wherein the ratio of OD260/OD280 is larger than 1.8, which indicates that the prepared RNA is purer and has no protein pollution.
2) Total RNA integrity detection: taking 1 μl of RNA sample, performing 1% agarose gel electrophoresis for 80V×20min, observing 5s rRNA, 18s rRNA and 28s rRNA bands of total RNA by using a gel imaging system, and if three bands are complete, proving that the total RNA extraction is complete.
Three, reverse transcription
The following solutions were prepared in RNASE FREE PCR tubes
（EasyScript First-Strand cDNA Synthesis SuperMix）
Incubating the 20. Mu.l of the reaction solution at 25℃for 10min;
Preserving the temperature at 42 ℃ for 30min;
The temperature is kept at 85 ℃ for 5s.
Fourth, quantitative PCR
1. Detecting sequence fragment size
Internal reference fragment: 18s-112bp.
The target fragment: tyrosinase-140bp;
| tyrosinase -F：5’ GGAACAAGCGAGTCGGATCT |
| tyrosinase -R：5’ GGAGTGGCTGCTTTTCTTCA |
| 18s-F：5’ CCTGGATACCGCAGCTAGGA |
| 18s-R：5’ GCGGCGCAATACGAATGCCCC |
。
2. the reaction system:
3. Reaction conditions:
95 ℃ for 5min; reading the plate at 95 ℃ for 15s and 60 ℃ for 32s, and performing 40 cycles;
Melting curve analysis: the temperature is 60-95 ℃.
Each sample was repeated 3 times.
Quantitative PCR instrument: ABI PRISM [ 7500 Sequence Detection System ].
Test results:
The QRT-PCR detection results are shown in FIG. 4, wherein Lane1 is day0, lane2 is day4, lane3 is day8, lane4 is day12, and Lane5 is day16. the expression level of tyrosinase is shown in FIG. 5.
The whitening efficacy test-the melanin skin model whitening test is carried out on the specific targeting tyrosinase prepared cosmetics prepared by the preparation method of the specific targeting tyrosinase, and the testing mechanism is Shanghai micro-spectrum detection technology group Co., ltd;
the test steps are as follows:
Preparing 24-well plates, adding 0.5 mL maintenance culture fluid to each well, and transferring the model into the marked 24-well plates; each test group required 3 models, and all 24 well plates with models were transferred to an incubator for incubation (37 ℃,5% co 2).
From the Day of model reception (Day 0), the Negative Control (NC), positive Control (PC) and sample groups were subjected to UVB irradiation (50 mJ/cm 2) daily, and the blank control group (BC) was not subjected to UVB irradiation. The model was changed every day. The positive control group (kojic acid, 500. Mu.g/mL) and the sample group were administered twice at Day3 and Day5, respectively, by surface administration, in a volume of 100. Mu.L.
After the model is continuously cultured for 7 days (Day 7), samples are collected and tested.
Apparent chromaticity test:
After the model cultivation is finished, apparent photographing is carried out by a camera. The specific photographing standard operation is as follows: (1) camera mode: manually; photographing parameters; setting: focal length=5.8 mm, aperture=f/8, aperture F22, shutter speed=1/80 s, iso=1600. (2) And placing the melanin skin model in the center of the color chart for photographing.
Apparent brightness (L x value) test:
and detecting the L-value of the model after the apparent chromaticity detection is finished. The specific detection operation is as follows: the model is placed on a flat and hard white plane, the stratum corneum is placed upwards, a detection hole of the color difference meter is vertically aligned with the surface of the model for detection, reading is repeated three times for each model, and an average value is taken as the reading of the L-value of a single model. The model after detection was placed in a clean EP tube for melanin content determination.
Melanin relative content test:
And detecting the melanin content of the model after the detection of the L value is finished. The detection operation is as follows:
(1) Placing the model in 1.5 mL EP tubes, marking, adding 1 mL PBS buffer solution into each tube, vibrating with a vortex oscillator to 3 min, centrifuging at low temperature and high speed with a centrifuge 2000 r/min, centrifuging with a centrifuge 10min, and discarding supernatant;
(2) Sequentially adding 200 mu L of distilled water, 500 mu L of absolute ethyl alcohol and 500 mu L of diethyl ether into the EP pipe in the step (1), fully and uniformly mixing, standing at room temperature for 20min, centrifuging at 3000 r/min for 5min, and discarding the supernatant;
(3) Adding 1 mL of 1 mol/L NaOH aqueous solution containing 10% DMSO, and heating in 80 ℃ water bath for 40 min;
(4) After the thermal incubation, 200 μl of supernatant was aspirated into the well corresponding to the well of the well 96 well plate with clear label, OD values were read at 405 nm and two replicates were tested on the well 96 plate for each model.
After the culture is finished according to the apparent chromaticity result, the picture of the melanin model shows that compared with the BC group, the apparent chromaticity of the NC group melanin model is deeper, which indicates that the melanin deposition on the surface of the model is more; the apparent chromaticity of the model for melanin was lighter for the PC and sample groups compared to NC group, indicating less melanin deposition on the model surface.
Apparent brightness L value test results:
the results of the L-value measurement of the model after the completion of the apparent chromaticity detection are shown in the following table and fig. 6.
| Group of | Average value of | SD | p-value |
| Blank Control (BC) | 77.73 | 1.75 | / |
| Negative Control (NC) | 63.63 | 2.24 | 0.021# |
| Positive Control (PC) | 78.16 | 0.58 | 0.012* |
| hTYR ASO-3 200nM | 77.73 | 1.26 | 0.020* |
When the statistical analysis is performed by using the t-test two-tailed test method, the significance of the NC group is represented by # compared with the BC group, p-value < 0.05 is represented by #, and p-value < 0.01 is represented by #. Compared with the NC group, the significance of the sample group and the PC group is expressed as the x, the p-value < 0.05 is expressed as the x, and the p-value < 0.01 is expressed as the x.
Based on the 3D melanin model apparent chromaticity L value test, compared with the BC group, the apparent brightness of the NC group is obviously reduced (p is less than 0.05), which proves that the UVB stimulation condition of the experiment is effective.
Compared with the NC group, the apparent brightness of the PC group is obviously improved (p is less than 0.01), which proves that the positive control of the experiment is effective.
The apparent brightness of the sample group was significantly improved compared to the NC group, with statistical differences (p < 0.05).
The magnitude of the absorbance (OD) reflects the relative amount of melanin and is measured as shown in the following table:
| Group of | Average OD value | SD | p-value |
| Blank Control (BC) | 0.1302 | 0.000 | / |
| Negative Control (NC) | 0.1860 | 0.002 | 0.000## |
| Positive Control (PC) | 0.1489 | 0.004 | 0.000** |
| hTYR ASO-3 200nM | 0.1371 | 0.007 | 0.001** |
When the statistical analysis is performed by using the t-test two-tailed test method, the significance of the NC group is represented by # compared with the BC group, p-value < 0.05 is represented by #, and p-value <0.01 is represented by #. Compared with the NC group, the significance of the sample group and the PC group is expressed as the x, the p-value < 0.05 is expressed as the x, and the p-value <0.01 is expressed as the x.
Based on Skinovo @ -Mela 3D melanin skin model, sample hTYR ASO-3 was able to increase apparent chromaticity and apparent brightness (L x values) and reduce melanin content with statistical differences (p < 0.05) compared to NC group after acting on the melanin skin model. It is shown that sample hTYR ASO-3 has whitening effect.
The stability test of the specific targeting tyrosinase provided by the invention comprises the following test steps:
The sample is converted into moving charged gaseous ion fragments by adopting ESI source ionization spray technology in a negative ion mode, and the fragments are separated and recorded according to mass-to-nuclear ratio (m/z) size.
The mass spectrum detection steps are as follows:
(1) Preparing a concentration sequence hTYR ASO of 100 uM for later use;
(2) Placing 500 ul nucleic acid solution into a water bath kettle at 60 ℃ for heating treatment by using a2 ml centrifuge tube with a well sealed spiral cover;
(3) Samples were taken weekly for 20 ul samples and diluted 6-fold with 100 ul ultrapure water;
(4) Mass spectrometry detects the conditions of lack of base, lack of thio and depurination, and each sampling is repeated for 3 times.
The results were as follows:
| Thio-lack (%) | Base deficiency (%) | Apurinic (%) | |
| As is | 2.00 | 0 | 0 |
| Heat treatment at 60 ℃ for 1 week | 2.17 | 0 | 0 |
| Heating at 60deg.C for 2 weeks | 5.89 | 0 | 0 |
| Heating at 60deg.C for 3 weeks | 5.48 | 0 | 0 |
| Heating at 60deg.C for 4 weeks | 7.23 | 0 | 2.16 |
。
From the mass spectrum detection results, it was found that as the heating treatment time became longer, part of the thio group was converted into oxo group. The ratio of successful oxo-conversion after 4 weeks of heat treatment increases from 2% to 7.23%; the proportion of missing bases does not rise as the heat treatment time becomes longer, and the sequence is still the full-length sequence; as the heat treatment time becomes longer and the depurination becomes stable, the aqueous solution becomes slightly acidic, and the depurination phenomenon occurs only under acidic conditions, and it is expected that the probe is not depurination by dissolution with a slightly basic TE buffer.
The liquid phase detection steps are as follows:
(1) Preparing a concentration sequence hTYR ASO of 100 uM for later use;
(2) Placing 500 ul nucleic acid solution into a water bath kettle at 60 ℃ for heating treatment by using a2 ml centrifuge tube with a well sealed spiral cover;
(3) Samples of 20 ul were taken weekly;
(4) The Waters 2795+PDA996 detector detects the liquid phase purity of the sequence.
| Purity (%) | |
| As is | 95.1 |
| Heat treatment at 60 ℃ for 1 week | 94.9 |
| Heating at 60deg.C for 2 weeks | 94.09 |
| Heating at 60deg.C for 3 weeks | 93.33 |
| Heating at 60deg.C for 4 weeks | 84.94 |
The high performance liquid chromatography detection result shows that the sample is basically stable after the heating treatment for 3 weeks, and the purity starts to be accelerated to be low in the fourth week; the purity deterioration is mainly due to the conversion of part of the thio-skeleton into oxo-skeleton; also a part of the depurination, which leads to a lower purity, can be avoided by dissolution with a more basic TE buffer.
Instrument device in high performance liquid chromatography detection: waters 2795+pda996 detector; chromatographic column: XBridge Oligonucleotide BEH Column,130A,3.5 μm,4.6 mm X50 mm; column temperature: 60 ℃; flow rate: 0.5 ml/min; detection wavelength: 260 nm; loading concentration: 100 uM; sample loading amount: 10 ul; mobile phase a: 0.1M TEAA,pH 7.2; mobile phase B: acetonitrile; the elution gradient was as follows:
| Time of | Flow rate (ml/min) | A% | B% | Detection wavelength (nm) |
| 0.00 | 0.5 | 100 | 0 | 260 |
| 0.50 | 0.5 | 100 | 0 | 260 |
| 0.51 | 0.5 | 90 | 10 | 260 |
| 22.00 | 0.5 | 80 | 20 | 260 |
| 22.01 | 0.5 | 0 | 100 | 260 |
| 24.99 | 0.5 | 50 | 50 | 260 |
| 25.00 | 0.5 | 100 | 0 | 260 |
。
The specific targeted tyrosinase provided by the application is also subjected to MTC (maximum tolerance concentration test), irritation (zebra fish neutrophil inhibition rate test), sensitization (zebra fish trypsin content test) and developmental toxicity (zebra fish embryo short-term developmental toxicity test), wherein the MTC (maximum tolerance concentration test) is 0.5nM, no death and deformity are observed, the specific targeted tyrosinase is not stimulated in the irritation (zebra fish neutrophil inhibition rate test), the specific targeted tyrosinase is not sensitized in the sensitization (zebra fish trypsin content test) at a concentration of 200nM, and the specific targeted tyrosinase is not developmental toxic in the fertility (zebra fish embryo short-term developmental toxicity test).
The main functions of the invention are as follows: the specific targeting tyrosinase provided by the invention can specifically target tyrosinase mRNA, and can inhibit the expression of tyrosinase, so that the production of melanin is inhibited for a long time, the whitening effect is achieved, the hTYR mRNA sequences are specifically degraded, the specificity is high, the safety is high, the special chemical modification is performed, the stability is high, and the degradation is difficult; without liposome delivery carrier, the liposome can self-pass through cell membrane and enter cell nucleus to act. The effective use concentration is low, the cost is controllable, and the use is convenient: can be used alone or in combination with other conventional cosmetics, and is suitable for various product forms such as ointment, spray or lyophilized powder.
In view of the above, after reading the present document, those skilled in the art should make various other corresponding changes without creative mental effort according to the technical scheme and the technical conception of the present invention, which are all within the scope of the present invention.
Claims (10)
1. The preparation method of the specific targeted tyrosinase is characterized by comprising the following preparation and synthesis routes:
，
Wherein the synthesis step comprises deprotection, coupling, oxidation and capping reaction steps;
Wherein the method comprises the steps of Is a solid phase synthetic linkage.
2. The method of claim 1, wherein the deprotecting step is of the formula:
，
Synthesizing the 3' -end first base of the oligonucleotide, selecting CPG linked with one base in A, G, C, T as a solid phase carrier, protecting the 5' OH of the base by a DMT protecting group, removing DMT of the first base by trichloroacetic acid before starting synthesis, and exposing active 5' OH; wherein the method comprises the steps of Is a solid phase synthetic linkage.
3. The method for preparing the specific targeted tyrosinase according to claim 1, wherein the coupling reaction formula is as follows:
，
Washing residual trichloroacetic acid on the solid phase carrier with anhydrous acetonitrile, adding tetrazole and corresponding phosphoramidite monomer, and reacting the phosphoramidite monomer activated by tetrazole with 5' OH on the solid phase carrier to form a phosphoramidite bond; wherein the method comprises the steps of Is a solid phase synthetic linkage.
4. The method for preparing the specific targeted tyrosinase according to claim 1, wherein the oxidation reaction formula is as follows:
Or (b)
，
I2 oxidizes trivalent phosphorus to pentavalent phosphorus, forming a phosphodiester bond.
5. The method for preparing the specific targeted tyrosinase according to claim 1, wherein the capping reaction formula is as follows:
，
The unreacted 5' OH is blocked with acetyl groups.
6. The method of claim 5, further comprising the step of removing the solid support according to the following reaction scheme:
。
7. The method for preparing the specific targeting tyrosinase according to claim 6, wherein the chemical reaction equation for removing the amino protecting group is as follows:
。
8. The method for preparing the specific targeted tyrosinase according to claim 7, further comprising the step of cleaving the phosphate backbone protecting group according to the following chemical reaction equation:
。
9. A specific targeting tyrosinase, characterized in that it is prepared by the method for preparing a specific targeting tyrosinase according to any one of claims 1 to 8.
10. The application of the specific targeting tyrosinase, which is characterized in that the specific targeting tyrosinase prepared by the preparation method of the specific targeting tyrosinase according to any one of claims 1 to 8 is applied to the preparation of cosmetics.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410832098.0A CN118388567A (en) | 2024-06-26 | 2024-06-26 | Specific targeted tyrosinase, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410832098.0A CN118388567A (en) | 2024-06-26 | 2024-06-26 | Specific targeted tyrosinase, preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118388567A true CN118388567A (en) | 2024-07-26 |
Family
ID=91994449
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410832098.0A Pending CN118388567A (en) | 2024-06-26 | 2024-06-26 | Specific targeted tyrosinase, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118388567A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1373768A (en) * | 1999-09-10 | 2002-10-09 | 杰龙公司 | Oligonucleotide N3'-P5' thiophosphoramidates, their synthesis and use |
| CN1375277A (en) * | 2001-03-20 | 2002-10-23 | 重庆富进生物医药有限公司 | Prevention of skin chromatosis with tyrosinase antesense oligodeoxynucleotide |
| CN108138181A (en) * | 2015-07-27 | 2018-06-08 | 奥利克斯医药有限公司 | Inhibit the RNA compounds of melanin generation |
| CN113667669A (en) * | 2021-08-06 | 2021-11-19 | 暨南大学 | Antisense oligonucleotide for inhibiting tyrosinase expression and application thereof |
-
2024
- 2024-06-26 CN CN202410832098.0A patent/CN118388567A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1373768A (en) * | 1999-09-10 | 2002-10-09 | 杰龙公司 | Oligonucleotide N3'-P5' thiophosphoramidates, their synthesis and use |
| CN1375277A (en) * | 2001-03-20 | 2002-10-23 | 重庆富进生物医药有限公司 | Prevention of skin chromatosis with tyrosinase antesense oligodeoxynucleotide |
| CN108138181A (en) * | 2015-07-27 | 2018-06-08 | 奥利克斯医药有限公司 | Inhibit the RNA compounds of melanin generation |
| CN113667669A (en) * | 2021-08-06 | 2021-11-19 | 暨南大学 | Antisense oligonucleotide for inhibiting tyrosinase expression and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 禹邦超等: "《酶工程（第三版）》", 31 January 2014, 华中师范大学出版社, pages: 135 * |
| 郑杰: "《肿瘤的细胞和分子生物学》", 30 April 2014, 军事医学科学出版社, pages: 308 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109641015A (en) | By removing tri-methylated increase human somatic cell nuclear transfer (SCNT) efficiency of histone H 3-lysine, and the method and composition of increase mankind NT-ESC derivative | |
| FR2558172A1 (en) | PROBE CONTAINING MODIFIED NUCLEIC ACID AND RECOGNITION BY SPECIFIC ANTIBODIES AND USE OF SUCH PROBE AND THESE SPECIFIC ANTIBODIES TO DETECT AND CHARACTERIZE A HOMOLOGATED DNA SEQUENCE | |
| CN111454895A (en) | Experimental method for repairing spinal cord by inducing microglia polarization through exosome | |
| CN1901838A (en) | Automated cytological sample classification | |
| CN112831497B (en) | Novel lncRNA and inhibitor, diagnostic reagent, medicament and application thereof | |
| CN118388567A (en) | Specific targeted tyrosinase, preparation method and application thereof | |
| CN112771158B (en) | Alkaline phosphatase composition and method for producing dephosphorylated nucleic acid and labeled nucleic acid | |
| JP7402429B2 (en) | Alkaline phosphatase composition and method for producing dephosphorylated nucleic acid and labeled nucleic acid | |
| CN115851947A (en) | Application of DAGLA in diagnosis and treatment of liver cancer | |
| CN114774417A (en) | MiRNA molecule for promoting schistosoma japonicum liver fibrosis, miRNA antagonist and application | |
| CN112159813B (en) | Nucleic acid aptamer specifically binding to ovarian serous adenocarcinoma cells and application thereof | |
| CN110951880B (en) | Application of reagent for detecting lncRNA marker of hypopharynx cancer in preparation of product for diagnosing hypopharynx cancer | |
| CN111154873B (en) | Molecular marker for detecting migration and invasion capacity of triple negative breast cancer cells and application thereof | |
| CN108165627B (en) | Molecular marker for diagnosing and treating pelvic cavity prolapse | |
| CN112877425A (en) | Application of reagent for detecting SNF5 gene expression in preparation of atherosclerosis diagnosis product | |
| CN109554369B (en) | Use of aptamers for recognition and binding of alkaline phosphatase heterodimers | |
| CN117643631B (en) | Use of inhibitors of CD151 and/or ITGB1 for the preparation of a formulation for inhibiting Porphyromonas gingivalis infection | |
| CN112656805A (en) | Application of substance for inhibiting YTHDF1 activity in preparation of product for preventing or treating gastric cancer | |
| CN111751532B (en) | Application of PIEZO1 protein as esophageal cancer marker | |
| CN116870025B (en) | Pulmonary fibrosis therapeutic cow milk exosome-siTGF-beta 1 medicine | |
| US20210046088A1 (en) | Methods and compositions for the diagnosis and treatment of endometriosis and endometriosis-related disorders | |
| CN104740649B (en) | Applications of the PLEKHA5 in tumour diagnostic reagent is prepared | |
| CN114032243B (en) | Aptamer specifically binding ciprofloxacin and application thereof | |
| CN115998729B (en) | Application of o-cresolphthalein in preparation of BLM helicase inhibitor or anticancer drug | |
| JP7402428B2 (en) | Method for evaluating quality of dephosphorylation reagent and method for detecting target nucleic acid |
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 |