CN110423754A - Aptamer and utilization SELEX screening technique and application with borax specific binding - Google Patents
Aptamer and utilization SELEX screening technique and application with borax specific binding Download PDFInfo
- Publication number
- CN110423754A CN110423754A CN201910371702.3A CN201910371702A CN110423754A CN 110423754 A CN110423754 A CN 110423754A CN 201910371702 A CN201910371702 A CN 201910371702A CN 110423754 A CN110423754 A CN 110423754A
- Authority
- CN
- China
- Prior art keywords
- borax
- aptamer
- bor
- specific binding
- aptamers
- 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
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 140
- 229910021538 borax Inorganic materials 0.000 title claims abstract description 125
- 239000004328 sodium tetraborate Substances 0.000 title claims abstract description 125
- 235000010339 sodium tetraborate Nutrition 0.000 title claims abstract description 125
- 238000000034 method Methods 0.000 title claims abstract description 43
- 238000012216 screening Methods 0.000 title claims abstract description 24
- 230000009870 specific binding Effects 0.000 title claims abstract description 16
- 108020004414 DNA Proteins 0.000 claims abstract description 24
- 238000001514 detection method Methods 0.000 claims abstract description 19
- 102000053602 DNA Human genes 0.000 claims abstract description 14
- 108090000790 Enzymes Proteins 0.000 claims abstract description 14
- 102000004190 Enzymes Human genes 0.000 claims abstract description 14
- 238000005516 engineering process Methods 0.000 claims abstract description 13
- 230000035945 sensitivity Effects 0.000 claims abstract description 12
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 11
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 10
- 239000002773 nucleotide Substances 0.000 claims abstract description 10
- 108020004682 Single-Stranded DNA Proteins 0.000 claims abstract description 8
- 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 claims abstract description 7
- 238000001179 sorption measurement Methods 0.000 claims abstract description 7
- 238000012163 sequencing technique Methods 0.000 claims description 14
- 239000011248 coating agent Substances 0.000 claims description 10
- 238000000576 coating method Methods 0.000 claims description 10
- 239000000427 antigen Substances 0.000 claims description 9
- 108091007433 antigens Proteins 0.000 claims description 9
- 102000036639 antigens Human genes 0.000 claims description 9
- 238000004458 analytical method Methods 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 238000000926 separation method Methods 0.000 claims description 8
- 230000000295 complement effect Effects 0.000 claims description 6
- 102000039446 nucleic acids Human genes 0.000 claims description 6
- 108020004707 nucleic acids Proteins 0.000 claims description 6
- 150000007523 nucleic acids Chemical class 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 238000000862 absorption spectrum Methods 0.000 claims description 4
- 238000002474 experimental method Methods 0.000 claims description 4
- 108091008104 nucleic acid aptamers Proteins 0.000 claims description 4
- 238000012512 characterization method Methods 0.000 claims description 3
- 238000013461 design Methods 0.000 claims description 3
- 238000000338 in vitro Methods 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 238000013518 transcription Methods 0.000 claims description 3
- 230000035897 transcription Effects 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- 241001349468 Elona Species 0.000 claims 2
- 238000010494 dissociation reaction Methods 0.000 abstract description 2
- 230000005593 dissociations Effects 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 description 30
- 239000000243 solution Substances 0.000 description 24
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 22
- 235000013305 food Nutrition 0.000 description 16
- 239000000853 adhesive Substances 0.000 description 12
- 230000001070 adhesive effect Effects 0.000 description 12
- 229960002685 biotin Drugs 0.000 description 11
- 235000020958 biotin Nutrition 0.000 description 11
- 239000011616 biotin Substances 0.000 description 11
- 238000005406 washing Methods 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 239000004327 boric acid Substances 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 230000008878 coupling Effects 0.000 description 8
- 238000010168 coupling process Methods 0.000 description 8
- 238000005859 coupling reaction Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 238000002965 ELISA Methods 0.000 description 7
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 7
- 239000007853 buffer solution Substances 0.000 description 7
- 239000008267 milk Substances 0.000 description 7
- 210000004080 milk Anatomy 0.000 description 7
- 235000013336 milk Nutrition 0.000 description 7
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 6
- 238000004925 denaturation Methods 0.000 description 6
- 230000036425 denaturation Effects 0.000 description 6
- 230000003321 amplification Effects 0.000 description 5
- 238000001816 cooling Methods 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 230000010355 oscillation Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 239000003053 toxin Substances 0.000 description 5
- 231100000765 toxin Toxicity 0.000 description 5
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000010926 purge Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000004064 recycling Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 238000002983 circular dichroism Methods 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 2
- 206010030113 Oedema Diseases 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 229910052796 boron Inorganic materials 0.000 description 2
- 150000001639 boron compounds Chemical class 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- -1 ethyl hexyl Chemical group 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 231100000636 lethal dose Toxicity 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- NNKVPIKMPCQWCG-UHFFFAOYSA-N methamidophos Chemical compound COP(N)(=O)SC NNKVPIKMPCQWCG-UHFFFAOYSA-N 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 2
- 229940043267 rhodamine b Drugs 0.000 description 2
- XWGJFPHUCFXLBL-UHFFFAOYSA-M rongalite Chemical compound [Na+].OCS([O-])=O XWGJFPHUCFXLBL-UHFFFAOYSA-M 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 239000013076 target substance Substances 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- GUYIZQZWDFCUTA-UHFFFAOYSA-N (pentadecachlorophthalocyaninato(2-))-copper Chemical compound [Cu+2].N1=C([N-]2)C3=C(Cl)C(Cl)=C(Cl)C(Cl)=C3C2=NC(C2=C(Cl)C(Cl)=C(Cl)C(Cl)=C22)=NC2=NC(C2=C(Cl)C(Cl)=C(Cl)C(Cl)=C22)=NC2=NC2=C(C(Cl)=C(C(Cl)=C3)Cl)C3=C1[N-]2 GUYIZQZWDFCUTA-UHFFFAOYSA-N 0.000 description 1
- XGIKILRODBEJIL-UHFFFAOYSA-N 1-(ethylamino)ethanol Chemical compound CCNC(C)O XGIKILRODBEJIL-UHFFFAOYSA-N 0.000 description 1
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 206010051290 Central nervous system lesion Diseases 0.000 description 1
- 244000163122 Curcuma domestica Species 0.000 description 1
- 235000003392 Curcuma domestica Nutrition 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 229910004835 Na2B4O7 Inorganic materials 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000005374 Poisoning Diseases 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 208000010513 Stupor Diseases 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000009102 absorption Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 231100000570 acute poisoning Toxicity 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000004596 appetite loss Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 125000005619 boric acid group Chemical group 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- HEDRZPFGACZZDS-UHFFFAOYSA-N chloroform Substances ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 235000003373 curcuma longa Nutrition 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 201000006549 dyspepsia Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 210000000750 endocrine system Anatomy 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000002117 illicit drug Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 208000019017 loss of appetite Diseases 0.000 description 1
- 235000021266 loss of appetite Nutrition 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 206010040882 skin lesion Diseases 0.000 description 1
- 231100000444 skin lesion Toxicity 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- GOLXNESZZPUPJE-UHFFFAOYSA-N spiromesifen Chemical compound CC1=CC(C)=CC(C)=C1C(C(O1)=O)=C(OC(=O)CC(C)(C)C)C11CCCC1 GOLXNESZZPUPJE-UHFFFAOYSA-N 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000007863 steatosis Effects 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 231100000816 toxic dose Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000008359 toxicosis Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 235000013976 turmeric Nutrition 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 208000016261 weight loss Diseases 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- 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
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to field of biomedicine technology, it discloses a kind of aptamer of and borax specific binding and utilizes SELEX screening technique and application, the aptamer is single stranded DNA, is made of 82 nucleotide, and nucleotide sequence is as shown in SEQ ID NO:1；Its secondary structure contains ring and stem outstanding, wherein Bor-B02 Gibbs free energy DG=-11.90.The a series of properties assessment such as aptamer specificity, affinity and sensitivity is carried out based on enzyme-linked oligonucleotides adsorption measurement, show Bor-B02 dissociation constant Kd=21.64 ± 2.732nM, and it can be specific in conjunction with borax, therefore the aptamer has the characteristics that high specific, high-affinity, can be used in the application of the subsequent quick detection kit to borax.
Description
Technical field
The invention belongs to the aptamer of field of biomedicine technology more particularly to a kind of specific binding with borax and
Utilize SELEX screening technique and application.
Background technique
Borax, generally writing Na2B4O7·10H2O is very important containing boron mineral and boron compound, usually colourless
The white powder of crystal, it is soluble easily in water.Borax can be used as detergent, cosmetics, insecticide, it can also be used to configure buffer solution and
Produce other boron compounds etc..The toxicity of borax is higher, is a kind of strong carcinogen, to the stomach of human body, kidney, liver, brain and
Skin lesion is very big, and the adult toxic dose of borax is 1-3 grams, and adult lethal dose is 15 grams, and baby's lethal dose is 2-3 grams.People
If body takes in excessive borax, the retention toxicosis of multi viscera can be caused.Long-term Excess free enthalpy borax, to human body reproduction, development
With the toxic influence of endocrine system.Short time takes in large dosage of borax, may cause acute poisoning, and less serious case causes dizzy, head
Bitterly, the symptoms such as loss of appetite, indigestion, weight loss；There are the poisoning manifestations such as vomiting, diarrhea, shock, stupor in serious person.
There is non-specific lesion in the visible stomach of the pathologic finding of borax poisoner, kidney, liver, brain and skin, mainly have liver it is congested,
Steatosis, liver cell cloudy swelling；Kidney is in diffusivity oedema, and glomerulus and renal tubule have damage；There is oedema in brain and lung.
The illegal producer and retailer are in order to increase the toughness, brittleness mouthfeel or extended shelf life of food, often in the processing of food
Illegal addition borax in the process.Since the strong carcinogenicity of borax and more virulent property, countries in the world generally forbid making an addition to borax
In food, prohibites in China " food hygiene law " and " food additives administration of health method " and added borax as food
Agent uses.
China not yet puts into effect the standard method for directly detecting borax in food, existing national standard " food at present
The measurement of boric acid in safe national standard-food " (GB 5009.275-2016) be by detect borax decomposition product boric acid
Content to calculate the content of borax in food indirectly.The existing detection method approved the most on the market is ethyl hexyl two
Alcohol-chloroform extraction turmeric colorimetric method.The method needs that borax is first changed into boric acid in acid condition, by detecting boric acid
Content to extrapolate the additive amount of borax indirectly.Due to the acidification in sample pre-treatments link and operation is filtered for multiple times, enables inspection
There is partial loss in the boric acid component surveyed in liquid, so that the result of the detection method has certain deviation, and detects knot
Fruit can only indicate there is boric acid in food, not necessarily directly be added to borax.In conclusion problem of the existing technology
Be: testing result has certain error, and method realizes the direct detection of borax in food.Therefore in order to guarantee numerous people group
The health of crowd fills up on the market the directly vacancy of detection borax method, establishes a kind of accurate and reliable, sensitive quick, extensive
The method of borax is particularly important in applicable direct detection food.
Aptamer is by index concentration Fas lignand system evolution technology (SyStematic evolution of
LigandS by exponential enrichment, SELEX) screening obtain can be big with metal ion, small molecule, biology
Molecule, or even the ssDNA or RNA molecule of entire cell high specific, high-affinity combination.Aptamer not only has antibody
The evident characteristics of molecule, it is all to be related to the diagnostic field of antibody, it can nearly all be replaced with aptamer, and be also equipped with
Oneself unique excellent properties, with target molecule range is wide, molecular mass is small, immunogenicity is low, is easy to chemical synthesis, transformation
The advantages that with label.Aptamer is in treatment new drug, medicament transport, cancer cell detection, bio-imaging, biological marker in recent years
Application in the fields of biomedicine such as the discovery of object, illicit drugs inspection, antiviral.Therefore, aptamer Technology application can be arrived
The direct detection of borax in food may be implemented in field of food detection.And the detection method established by the technology is without more
Cumbersome sample pretreatment step, can reduce the loss of borax, so that testing result is more accurate reliable.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of with the borax aptamer of specific binding and
Utilize SELEX screening technique and application.
The invention is realized in this way a kind of aptamer with borax specific binding, described specific with borax
In conjunction with aptamer nucleotide sequence as shown in SEQ ID NO:1.
Further, the secondary structure with the aptamer of borax specific binding has ring outstanding and stem, lucky
Buss free energy DG=-11.90.
Further, the aptamer with borax specific binding is based on enzyme-linked oligonucleotides adsorption measurement
ELONA carries out its specificity, affinity and sensitive nature assessment.
Another object of the present invention is to provide the screening sides described in one kind with the aptamer of borax specific binding
Method, the screening technique of the aptamer of the specific binding with borax the following steps are included:
The first step, screening, clone, separation and the sequencing of borax specific nucleic acid aptamers；
Second step, aptamer single stranded DNA secondary structure characterization；
Third step, the preparation of borax coating antigen；
4th step, specificity, affinity and the sensitivity to borax of aptamer；
5th step, the truncation experimental analysis of aptamer.
Further, the first step specifically includes: going out the core that can be specifically bound with borax using SELEX technology screening
Sour aptamers group, design primer carry out PCR amplification, clone, separation and sequencing；It is carried out using enzyme-linked oligonucleotides adsorption experiment
Verifying, obtain aptamers Bor-B02, it can high-affinity, high specific combination borax；The adapter-primer sequence of aptamers
Are as follows: Aptamer Fw:GACATATTCAGTCTGACAGC；Reverse complementary sequence: CGCTGTCAGACTGAATATGTC；
AptamerRv:GCTAGACGATATTCGTCCATC, reverse complementary sequence: GATGGACGAATATCGTCTAGC；It obtains positive single
Clone carries out nucleotide sequencing, and sequencing result shows the aptamer specifically bound with borax by 82 nucleotide groups
At sequence (5 ' ends to 3 ' ends) are as follows:
GCTAGACGATATTCGTCCATCTCCCGTGCATGCGAGACCGTGAGTTGGACTGACCCGCTTCCGCTGTC
AGACTGAATATGTC。
Further, the second step specifically includes: the aptamer specifically bound using MFOLD software pair with borax
Bor-B02 single strand dna carries out secondary structure prediction；Secondary structure has ring outstanding and stem, Gibbs free energy DG=-
11.90。
Further, the third step specifically includes: by chemical method by borax molecule and BSA coupling to obtain borax
Coating antigen judges the preparation situation of coating antigen by ultra-violet absorption spectrum, and borax is successfully coupled with BSA as the result is shown；
4th step specifically includes: according to the sequence of aptamer Bor-B02, with in-vitro transcription method synthesising biological
The aptamer of element label, establishes enzyme-linked oligonucleotide detection method, to the specificity of aptamer, affinity and its right
The sensitivity of borax is detected.
Further, the 5th step specifically includes: being truncated aptamer Bor-B02 with true by ELONA method
Determine best combination site；Based on the secondary structure of aptamer Bor-B02 prediction, aptamers Bor-B02 is truncated.
Another object of the present invention is to provide the aptamers described in one kind with borax specific binding to identify boron
Application in sand kit.
In conclusion advantages of the present invention and good effect are as follows: compared with existing borax detection technique on the market, utilize
The aptamer Bor-B02 that SELEX technology is filtered out can high-affinity, high specific identification and combine borax, because
The direct detection of borax in food may be implemented in this subsequent detection technique based on aptamer；Aptamer Bor-B02
Specificity, affinity and sensitivity identification, it is ensured that the accuracy of the testing result of borax remaining in food.Based on core
The detection method of sour aptamers is not necessarily to cumbersome sample pre-treatments step, reduces the loss of borax in sample liquid, improves testing result
Reliability.Therefore the present invention has filled up at present the direct blank of detection borax method both at home and abroad, to preventing borax in food
It uses, reduces borax and be of great significance to the infringement of human body.
Detailed description of the invention
Fig. 1 is the secondary structure schematic diagram of aptamer provided in an embodiment of the present invention.
Fig. 2 is circular dichroism detector provided in an embodiment of the present invention to K+ concentration and aptamer Bor-B02 structural relation
Analysis result.
Fig. 3 is the uv absorption spectra of boric acid-BSA conjugate provided in an embodiment of the present invention.
Fig. 4 is that be ELONA method provided in an embodiment of the present invention analyze the specificity of aptamer Bor-B02, in figure:
Blank control: defatted milk；Negative control is non-target substance: rhodamine B, acephatemet, sodium formaldehyde sulfoxylate, boric acid；Positive group: borax.
Fig. 5 is affinity analysis of the ELONA method provided in an embodiment of the present invention to aptamer Bor-B02, and nucleic acid is suitable
The affinity of the combination of ligand Bor-B02 and borax is indicated with Kd value.
Fig. 6 is provided in an embodiment of the present invention to be divided based on ELONA method aptamer Bor-B02 borax sensitivity
It analyses, in figure: blank control: defatted milk；Positive group: borax.
Fig. 7 is the secondary structure figure of the aptamer Bor-B02 after different loci provided in an embodiment of the present invention truncates.
Fig. 8 is truncation experimental analysis of the ELONA method provided in an embodiment of the present invention to aptamer Bor-B02, in figure:
Blank control CK1: defatted milk；Blank control CK2: biotin；Positive group: being marked with the aptamers Bor-B02 and mark of biotin
Note has truncation aptamers Bor-B0471, Bor-B0449, Bor-B2471, Bor-B0435, Bor-B2449, Bor- of biotin
B3771、Bor-B0418、Bor-B2435、Bor-B3749、Bor-B4971。
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Application principle of the invention is explained in detail with reference to the accompanying drawing.
The aptamer provided in an embodiment of the present invention for obtaining specifically binding with borax using SELEX technology screening
Bor-B02 method the following steps are included:
The first step, screening, clone, separation and the sequencing of borax specific nucleic acid aptamers；
Go out the aptamer group that can be specifically bound with borax using SELEX technology screening, design primer carries out
PCR amplification, clone, separation and sequencing.It is verified using enzyme-linked oligonucleotides adsorption experiment (ELONA), obtains aptamers
Bor-B02, it can high-affinity, high specific combination borax.The adapter-primer sequence of aptamers are as follows: Aptamer Fw:
GACATATTCAGTCTGACAGC；Reverse complementary sequence: CGCTGTCAGACTGAATATGTC；Aptamer Rv:
GCTAGACGATATTCGTCCATC, reverse complementary sequence: GATGGACGAATATCGTCTAGC；Obtained positive monoclonal carries out core
Nucleotide sequence measurement, sequencing result show that the aptamer specifically bound with borax is made of 82 nucleotide, sequence
(5 ' ends to 3 ' ends) are as follows:
GCTAGACGATATTCGTCCATCTCCCGTGCATGCGAGACCGTGAGTTGGACTGACCCGCTTCCGCTGTCA
GACTGAATATGTC
Second step, aptamer single stranded DNA secondary structure characterization
Do you use MFOLD software (http://mfold.rna.albany.edu/ q=mfold/DNA-Folding-Form)
Secondary structure prediction is carried out to the aptamer Bor-B02 single strand dna specifically bound with borax.The result shows that
Secondary structure has ring outstanding and stem, and Gibbs free energy DG=-11.90 shows structure stability with higher；Its
Secondary structure is shown in Fig. 1.
Third step, the preparation of borax coating antigen
According to the structural analysis to borax molecule, borax molecule can not be tied by the group of itself with ELISA Plate
It closes.Therefore it needs to be coupled borax molecule and BSA by chemical method to obtain the coating antigen of borax, can be used for subsequent enzyme-linked
In oligonucleotides adsorption measurement.The preparation situation of coating antigen is judged by ultra-violet absorption spectrum, as the result is shown borax and BSA at
Function coupling.
4th step, specificity, affinity and the sensitivity to borax of aptamer
According to the sequence of aptamer Bor-B02, the aptamer marked with in-vitro transcription method synthesizing biotinylated,
Establish a kind of novel enzyme-linked oligonucleotide detection method, by the method to the specificity of aptamer, affinity and
It detects the sensitivity of borax；The results show that Bor-B02 dissociation constant Kd=21.64 ± 2.732nM, and Bor-
B02 has very high specificity, can detect that the minimum concentration of borax is not 20ng/mL.
5th step, the truncation experimental analysis of aptamer
Aptamer Bor-B02 is truncated by ELONA method to determine best combination site.It is adapted to based on nucleic acid
The secondary structure (Fig. 1) of body Bor-B02 prediction, truncates aptamers Bor-B02, the following table of sequence after truncation, second level knot
Structure is as shown in Figure 7；The results show that the segment after truncating cannot identify target molecule, therefore, aptamer Bor-B02 shows it
Complete three-dimensional structure is capable of the identification target molecule of high-affinity.
Application principle of the invention is further described combined with specific embodiments below.
Embodiment 1: the screening of borax aptamer
One, the coupling of aglucon (borax) and matrix (Ago-Gel 6B)
1, in sintered glass filter (more reciprocal of duty cycle G3) with the freeze-dried powder Ago-Gel of suspension 1g in 3mL distilled water
6B；
2, the coupling buffer solution (carbonate buffer of 0.05M, pH9.6 of 200mL is used in sintered glass filter immediately
Liquid) washing matrix 1 hour；-
3, with the aglucon of the coupling buffer solution dissolution 6g of 6mL, make its ultimate density 1mg/mL；
It 4, is that 1mg/mL has dissolved the buffer solution of aglucon 1:2 ratio by volume by the concentration in step 2 matrix and step 3
Example mixing is handled 16 hours under 35 DEG C -40 DEG C of water bath condition, and 37 DEG C of shaking tables are stayed overnight；
5, under conditions of 40 DEG C -50 DEG C, with the superfluous group of the ethylaminoethanol of 1M closing, at least 4 hours or overnight；
6, surplus aglucon is washed with coupling buffer, 4000rpm is centrifuged 2 minutes, draws supernatant；With ultrapure water (each 7-
8mL) clean 3 times；And then it uses and contains 0.1M NaHCO3, 0.5M NaCl pH8.0 solution clean 3-4 times；Then with containing
Have 0.1M NaCl, 0.1M acetate pH 4.0 solution clean 3-4 times；It is finally 20% ethyl alcohol with mass percent concentration
6mL is saved, and is placed in 4 DEG C of refrigerators, and sealed membrane sealing is vertical to place.
Two, the PCR of aptamer library (ssDNA)
The ssDNA aptamers library synthesized using TaKaRa company；
1,94 DEG C of preheating PCR instruments；
2, by 3 μ L ssDNA, 30.6 μ L deionized waters, 5 μ L10 × Buffer, 3 μ LMgCl2, 4 μ L dNTP mixtures it is (each
2.5 μM), 2 μ L forward direction amplimers, the reversed amplimer of 2 μ L and 0.4 μ LTaq enzyme centrifuge tube reacted；Forward direction amplification is drawn
Object sequence is 5 '-GACATATTCAGTCTGACAGC-3 ', and reversed amplimer sequence is 5 '-
GCTAGACGATATTCGTCCATC-3’。
3, it is expanded in PCR instrument by following procedure
(1) initial denaturation
94 DEG C 5 minutes；
(2) 30 circulations
94 DEG C 45 seconds
58 DEG C 30 seconds
72 DEG C 30 seconds；
(3) it expands afterwards
72 DEG C 7 minutes.
Three, the purifying in ligand library, loading and elution
1, the purifying in ligand library
(1) directly 1:1 ratio is mixed by volume with the solution in plastic recovery kit for aptamer library PCR product
It closes, carries out DNA fragmentation recycling；
(2) DNA fragmentation recycling after, 95 DEG C water-bath 10 minutes, ice bath 10 minutes；By this denaturation treatment, become double-stranded DNA
At single stranded DNA.
2, affinity chromatography
(1) matrix that was coupled being eluted with coupling buffer: drawing upper solution, coupling buffer is added to mix to 6mL,
Centrifugation, discards supernatant liquid, this step is repeated 3 times；
(2) single stranded DNA in step 1 is added in the matrix being coupled, is incubated in 37 DEG C and 40rpm softly rotates 2
Hour；
(3) aforementioned sample is added into chromatographic column, with the ultrapure water pillar of 2-3 column volume；
(4) linear ladder then is carried out with the elution buffer (0.1M NaCl, 0.1M acetate, pH4.0) of 3-4 column volume
Degree elution, collects elution fraction；
(5) elution fraction mixes in equal volume with sol solution, is dissolved after recycling with TE solution, obtains Selex solution.
Four, PCR optimization and massive amplification aptamer
Using above-mentioned Selex solution as template, operate as follows:
1,94 DEG C of preheating PCR instruments；
2, by 5 μ L templates, 28.6 μ L deionized waters, 5 μ L10 × Buffer, 3 μ LMgCl2, 4 μ L dNTP mixtures it is (each
2.5 μM), 2 μ L forward direction amplimers, the reversed amplimer of 2 μ L, 0.4 μ LTaq enzyme, reacted in PCR centrifuge tube.It is positive
Amplimer sequence is 5 '-GACATATTCAGTCTGACAGC-3 ', and reversed amplimer sequence is 5 '-
GCTAGACGATATTCGTCCATC-3’。
3, it is expanded in PCR instrument by following procedure:
(1) initial denaturation
94 DEG C 5 minutes；
(2) 32 circulations:
94 DEG C 45 seconds
58 DEG C 30 seconds
72 DEG C 30 seconds；
(3) it expands afterwards
72 DEG C 7 minutes；
4, PCR product after circulation terminates, is stripped recycling with the DNA product purification kit of TIANGEN company, is walked
It is rapid as follows:
(1) PCR product and isometric colloidal sol buffer are mixed by inversion, mixed liquor are then transferred to centrifugal purification column,
It is stored at room temperature 5 minutes, makes DNA sufficiently in conjunction with pellosil, 12000rpm is centrifuged 1 minute, outwells the waste liquid in collecting pipe；
(2) rinsing liquid (containing ethyl alcohol) of 700 μ L is added in centrifugal purification column, 12000rpm is centrifuged 1 minute, outwells collection
Waste liquid in pipe；
(3) step (2) are repeated；
(4) 12000rpm is centrifuged 3 minutes；
(5) centrifugal purification column is placed in new centrifuge tube；
(6) 30 μ L ultrapure waters are added, stand 5 minutes at room temperature；
(7) 12000rpm is centrifuged 1 minute, and tube bottom solution is the PCR product of the aptamer purified.
Embodiment 2: the clone of aptamer, separation and sequencing and the prediction of single stranded DNA secondary structure
One, the preparation of bacillus coli DH 5 alpha competent cell
1, the single DH5 α bacterium colony of picking is inoculated in 3mL LB culture medium not with ampicillin, 37 DEG C of overnight incubations,
Next day takes above-mentioned bacterium solution, and 1:100 is inoculated in 50mL LB liquid medium in proportion, and 37 DEG C vibrate 2 minutes；When OD600 value reaches
When to 0.35, bacterial cultures is harvested；
2, bacterial cultures is transferred in the sterile polypropylene tube of 50mL pre-cooling, places 10 minutes on ice, makes to train
It is cooling to support object；
3,4000rpm is centrifuged 10 minutes at 4 DEG C, discards culture solution, and pipe is inverted l/min so that remaining culture solution
It flows to end；
4, respectively add the 0.1mM CaCl of 150 μ L ice pre-cooling2Solution merges two and manages, and ice bath 10 minutes；
5,4000rpm is centrifuged 10 minutes at 4 DEG C, discards supernatant liquid, and pipe is inverted l/min so that residual liquid stream
To the greatest extent；
6, the 0.1M CaCl of 800 μ L ice pre-cooling is first added2Cell is resuspended in solution, adds the sweet of the 75% of 25 μ L pre-cooling
Oil, it is spare in -80 DEG C of storages later.
Two, connection and the conversion of connection product
1,0.5 μ LTakarapMD19-T Simple carrier, 4.5 μ L aptamer PCR are added in microcentrifugal tube
The ligase buffer mixture of product and 5 μ L；
2, it reacts 3 minutes for 16 DEG C；
3, full dose (10 μ L) is added into 100 μ L DH5 α competent cells, places 30 minutes in ice；
4,42 DEG C after heating 90 seconds, then place 1 minute in ice；
5, the LB culture medium 890 μ L for being added that 37 DEG C of warm bath cross, 37 DEG C slow oscillation culture 60 minutes；
6, take 200 μ L be coated on containing X-Gal, IPTG, ampicillin LB culture medium on, 37 DEG C culture 16 hours with
Form single colonie.
Three, the colony screening of aptamer, separation and sequencing and single stranded DNA secondary structure prediction
The single bacterium of the above-mentioned white of picking is fallen in LB culture medium with ampicillin, 37 DEG C slow oscillation culture 4 hours,
Carry out PCR amplification；The amplification condition of amplimer and amplification condition with aforementioned aptamer.Positive gram will confirmed through PCR
After grand progress plasmid extraction, nucleosides is carried out with the full-automatic nucleotide sequencing instrument of U.S. Applied BioSyStemS3730A
The measurement of acid sequence；Structure shows its sequence of aptamer specifically bound with borax from 5 ' ends to 3 ' ends are as follows:
GCTAGACGATATTCGTCCATCTCCCGTGCATGCGAGACCGTGAGTTGGACTGACCCGCTTCCGCTGTCA
GACTGAATATGTC
The sequence length of the aptamer Bor-B02 specifically bound with borax: 82 bases, sequence type: nucleic acid,
Chain number: single-stranded, topology: straight-chain, sequence type: ssDNA.
It is 26 DEG C, Na that temperature, which is arranged, by MFOLD software+Concentration is 150mM, Mg2+Concentration be 1mM (http: //
Mfold.rna.albany.edu/ q=mfold/DNA-Folding-Form) and QGRS mapping (http: //
BioinformaticS.ramapo.edu/QGRS/analyze.php) to the aptamer Bor- specifically bound with borax
B02 single strand dna carries out secondary structure prediction.The result shows that aptamers contain ring and stem outstanding, Bor-B02 gibbs
Free energy DG=-11.90；Structure stability with higher (see Fig. 1).
Embodiment 3: circular dichroism detector (CD) is to K+Concentration is probed into aptamer Bor-B02 structural relation
1, by aptamer ddH2O is diluted to 20 μM, after 95 DEG C of denaturation 30s, is cooled to 25 DEG C rapidly.Again
The aptamer after above-mentioned denaturation is diluted to 2.5 μ with the KCl solution of various concentration (0,10,20,40,60,80,100mM)
M。
2, aptamer is diluted to 20 μM with 10mM PBS, after 95 DEG C of denaturation 30s, is cooled to 25 rapidly
DEG C, then 2.5 μM are diluted to 10mM PBS.
3, temperature setting is 25 DEG C, is detected with circular dichroism spectrometer, wherein the scanning wavelength set is 220-340nm；
Scanning speed is 100nm/min；Reaction time is 1s.
The result shows that: in various concentration K+Under environment, the positivity band of the CD map of aptamers Bor-B02 is both present in
Near 280nm, negativity band is both present near 245nm, is intersected near 260nm with b axis, complies with standard the CD figure of DNA
Spectrum.And the fluctuating range of each curve is unobvious, therefore aptamer Bor-B02 can be stabilized simultaneously in KCl solution
And it will not influence its secondary structure (see Fig. 2).
Embodiment 4: borax-BSA conjugate preparation
1,2mg borax is dissolved with 0.2mL dimethylformamide (DMF), A liquid is made.
2,5mg BSA, 3mg EDPC addition 25mL ultrapure water are dissolved, B liquid is made.
3, A liquid is gradually added drop-wise in B liquid, stirring while adding, 1mg EDPC after 10min, in room temperature, the item that pH is 5.5
Continue to stir 18h under part.
4, it with PBS solution dialysis 3d, changes the liquid once daily.After having dialysed, packing freeze-drying is saved.
The result shows that: with borax, BSA characteristic peak compared with, borax-BSA compound in 290nm, 434nm, 471nm,
There is different characteristic ultraviolet absorptions at 509nm, 549nm, shows that borax and BSA are coupled successfully (see Fig. 3).
Embodiment 5: specificity, affinity and the sensitivity to borax of aptamer Bor-B02
One, aptamer Bor-B02 specific detection
1, ELONA method
It is improved on the basis of traditional ELISA method, antibody is replaced with the aptamers screened, using life
Object element-Avidin amplification system is to detect sample to be tested.
(1) coating of toxin
9.6 carbonate buffer solution of 0.05M pH and borax volume ratio are 1:1 mixing, the final concentration of 1 μ g/mL of borax.Often
100 μ L of hole is added in ELISA Plate, is sealed with adhesive sticker, in being incubated for 2 hours on 37 DEG C, 100rpm oscillator, abandoning after being incubated for
The liquid in hole is removed, 200 μ L PBST cleaning solutions are added in every hole, oscillation washing 3 times on Yu Shuiping constant-temperature table, 2 points of washing every time
It is patted dry after clock on clean blotting paper.Blank control and negative control (blank control: defatted milk are set up simultaneously；Negative control
For non-target substance: rhodamine B, acephatemet, sodium formaldehyde sulfoxylate, boric acid；Positive group: borax.Concentration is consistent, and method is same as above).
(2) it closes
In the ELISA Plate for being coated with borax toxin, 5% defatted milk of 100 μ L is added in each hole, is sealed with adhesive sticker, it
Afterwards, in being incubated for 2 hours on 37 DEG C, 100rpm oscillator, after closing, liquid in hole is discarded, repeats the purge step in (1)
Suddenly.
(3) plus the aptamer with biotin labeling is incubated for
It screens the aptamer Bor-B02 for obtaining to combine with borax toxin and is sent to the conjunction of Shanghai Sheng Gong biotech firm
At, and Bor-B02 is marked with biotin (Biotin).In use, first carrying out of short duration centrifugation, make the nucleic acid of labeled biotin
Aptamers are all gathered in test tube bottom.It is with aqua sterilisa that the aptamer of labeled biotin is sufficiently molten according to explanation
Solution is 10 at concentration-4The storing solution of M can be packed as aliquot in order to avoid freeze thawing repeatedly.
The aptamer Bor-B02 of labeled biotin is diluted to the working concentration of 100nM with 1 × PBS, then,
Accordingly 100 μ L are added in each hole, after adhesive sticker or sealed membrane sealing, in being incubated for 2 hours on 37 DEG C, 100rpm oscillator,
After incubation, liquid in hole is discarded, repeats the washing step in (1).
(4) enzyme conjugate is incubated for
The horseradish peroxidase conjugate that 100 μ L indicate Streptavidin is added in each hole, it is then, close with adhesive sticker
Envelope in being incubated for 1 hour on 37 DEG C, 100rpm oscillator, after incubation, discards liquid in hole, repeats the washing in (1) later
Step.
(5) it develops the color
100 μ L of color developing agent TMB solution is added in every hole, is protected from light colour developing 20 minutes at 37 DEG C later.
(6) it terminates
Finally, the terminate liquid (2M sulfuric acid) of 50 μ L is added into every hole, and to make within 10 minutes of reaction terminating
Absorbance value of each hole at 450nm is detected with microplate reader, obtains OD450nm.
The results show that aptamers Bor-B02 can and borax specificity combination (see Fig. 4).
Two, the affinity K of aptamer Bor-B02dValue calculates
1,9.6 carbonate buffer solution of 0.05M pH and borax volume ratio are 1:1 mixing, the final concentration of 1 μ g/mL of borax.
Every 100 μ L of hole is added in ELISA Plate, is sealed with adhesive sticker, in being incubated for 2 hours on 37 DEG C, 150rpm oscillator, after being incubated for
Discard liquid in hole, every hole adds 200 μ L of cleaning solution, on horizontal shaker oscillation washing 3 times, 2 minutes every time, similarly every time
It is to pat dry on net blotting paper；
2, in the ELISA Plate for being coated with borax toxin, 5% defatted milk of 100 μ L is added in each hole, is sealed with adhesive sticker,
Later, in being incubated for 2 hours on 37 DEG C, 100rpm oscillator, after closing, liquid in hole is discarded, repeats the purge step in 1
Suddenly；
3, by the aptamers of biotin labeling with 1 × PBS be diluted to 1nM, 5nM, 10nM, 20nM, 50nM, 80nM,
100nM, 150nM, 300nM, 500nM, each hole add 100 μ L, are sealed with adhesive sticker or sealed membrane, vibrate in 37 DEG C, 100rpm
It is incubated for 2 hours on device, liquid in hole is discarded after being incubated for, repeat the washing step in 1；
3,100 μ L horseradish peroxidase conjugates are added in every hole, are sealed with adhesive sticker, are vibrated in 37 DEG C, 150rpm
It is incubated for 1 hour on device, discards liquid in hole, repeat the washing step in 1；
4,100 μ L of TMB color developing agent is added in every hole, and colour developing 20 minutes is protected from light in 37 DEG C；
5, add 50 μ L terminate liquids (2M sulfuric acid), and surveyed at each hole 450nm and inhaled with microplate reader within reaction terminating 10 minutes
Shading value OD450nm；
The result shows that the K of aptamer Bor-B02d=21.64 ± 2.732nM (see Fig. 5).
Four, detection of the aptamer Bor-B02 to borax sensitivity
1,9.6 carbonate buffer solution of 0.05M pH and borax volume ratio are 1:1 mixing, make the final concentration of 1ng/ of borax
ML, 5ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 60ng/mL, 80ng/mL, 100ng/mL.Every 100 μ L of hole is added to enzyme
It in target, is sealed with adhesive sticker, in being incubated for 2 hours on 37 DEG C, 150rpm oscillator, liquid in hole, every hole is discarded after being incubated for
Add 200 μ L of cleaning solution, in oscillation washing 3 times on horizontal shaker, 2 minutes every time, to be similarly net blotting paper every time
On pat dry；
2, in the ELISA Plate for being coated with borax toxin, 5% defatted milk of 100 μ L is added in each hole, is sealed with adhesive sticker,
Later, in being incubated for 2 hours on 37 DEG C, 100rpm oscillator, after closing, liquid in hole is discarded, repeats the purge step in 1
Suddenly；
3, the aptamers of biotin labeling are diluted to 100nM with 1 × PBS, each hole adds 100 μ L, with adhesive sticker or envelope
Membrana oralis sealing discards liquid in hole in being incubated for 2 hours on 37 DEG C, 100rpm oscillator after being incubated for, repeat the purge step in 1
Suddenly；
4,100 μ L horseradish peroxidase conjugates are added in every hole, are sealed with adhesive sticker, are vibrated in 37 DEG C, 150rpm
It is incubated for 1 hour on device, discards liquid in hole, repeat the washing step in 1；
5,100 μ L of TMB color developing agent is added in every hole, and colour developing 20 minutes is protected from light in 37 DEG C；
6, add 50 μ L terminate liquids (2M sulfuric acid), and surveyed at each hole 450nm and inhaled with microplate reader within reaction terminating 10 minutes
Shading value OD450nm；
The result shows that aptamer Bor-B02 detects that the minimum concentration of borax is not 20ng/mL (see Fig. 6).
Embodiment 6: the truncation experimental analysis of aptamer Bor-B02
In order to reduce the cost of nucleic acid aptamers, to aptamer Bor-B02 be based on its secondary structural features into
Row truncates experiment and gropes.Sequence after aptamer truncates see the table below, and secondary structure is as shown in Figure 7.Ten are truncated respectively
Aptamer segment and full length fragment afterwards carries out ELONA method and is verified, remaining operation is same as above.
The result shows that aptamer Bor-B02 truncate after segment Bor-B0471, Bor-B0449, Bor-B2471,
Although Bor-B0435, Bor-B3771, Bor-B0418 can identify borax molecule, its specific recognition capability is obviously low
In the aptamer of full length fragment.Thus judge, multiple structural regions of aptamer take part in itself and borax molecule
In conjunction with the removal in part-structure region may will affect the specific recognition capability of aptamer.Therefore, in order to guarantee nucleic acid
High specific, the high-affinity of aptamers, should select the aptamer of full length fragment to conduct further research.(Fig. 8).
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>Kunming University of Science and Technology
<120>aptamer specifically bound with borax and utilization SELEX screening technique and application
<130> 2018
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 82
<212> DNA
<213>artificial synthesized (artificial)
<400> 1
gctagacgat attcgtccat ctcccgtgca tgcgagaccg tgagttggac tgacccgctt 60
ccgctgtcag actgaatatg tc 82
Claims (9)
1. a kind of aptamer with borax specific binding, which is characterized in that the nucleic acid with borax specific binding
Aptamers nucleotide sequence is as shown in SEQ ID NO:1.
2. the aptamer specifically bound as described in claim 1 with borax, which is characterized in that described special with borax
Property combine aptamer secondary structure have ring outstanding and stem, Gibbs free energy DG=-11.90.
3. the aptamer specifically bound as described in claim 1 with borax, which is characterized in that described special with borax
Property the aptamer that combines be based on enzyme-linked oligonucleotides adsorption measurement ELONA, carry out its specificity, affinity and sensitivity
Property assessment.
4. a kind of screening technique with the aptamer of borax specific binding as described in claim 1, which is characterized in that institute
State with borax specific binding aptamer screening technique the following steps are included:
The first step, screening, clone, separation and the sequencing of borax specific nucleic acid aptamers；
Second step, aptamer single stranded DNA secondary structure characterization；
Third step, the preparation of borax coating antigen；
4th step, specificity, affinity and the sensitivity to borax of aptamer；
5th step, the truncation experimental analysis of aptamer.
5. the screening technique of the aptamer specifically bound as claimed in claim 4 with borax, which is characterized in that described
The first step specifically includes: going out the aptamer group that can be specifically bound with borax using SELEX technology screening, design is drawn
Object carries out PCR amplification, clone, separation and sequencing；It is verified using enzyme-linked oligonucleotides adsorption experiment, obtains aptamers Bor-
B02；The adapter-primer sequence of aptamers are as follows: Aptamer Fw:GACATATTCAGTCTGACAGC；Reverse complementary sequence:
CGCTGTCAGACTGAATATGTC；Aptamer Rv:GCTAGACGATATTCGTCCATC, reverse complementary sequence:
GATGGACGAATATCGTCTAGC；Obtained positive monoclonal carries out nucleotide sequencing, and sequencing result shows special with borax
Property the aptamer that combines be made of 82 nucleotide, sequence (5 ' end to 3 ' end) are as follows:
GCTAGACGATATTCGTCCATCTCCCGTGCATGCGAGACCGTGAGTTGGACTGACCCGCTTCCGCTGTCAGAC
TGAATATGTC。
6. the screening technique of the aptamer specifically bound as claimed in claim 4 with borax, which is characterized in that described
Second step specifically includes: the aptamer Bor-B02 single strand dna specifically bound using MFOLD software pair with borax
Carry out secondary structure prediction；Secondary structure has ring outstanding and stem, Gibbs free energy DG=-11.90.
7. the screening technique of the aptamer specifically bound as claimed in claim 4 with borax, which is characterized in that described
Third step specifically includes: being coupled to obtain the coating antigen of borax for borax molecule and BSA by chemical method, passes through UV absorption
Spectrum judges the preparation situation of coating antigen, and borax is successfully coupled with BSA as the result is shown；
4th step specifically includes: according to the sequence of aptamer Bor-B02, with in-vitro transcription method synthesizing biotinylated mark
The aptamer of note establishes enzyme-linked oligonucleotide detection method, to the specificity of aptamer, affinity and its to borax
Sensitivity detected.
8. the screening technique of the aptamer specifically bound as claimed in claim 4 with borax, which is characterized in that described
5th step specifically includes: is truncated to aptamer Bor-B02 by ELONA method to determine best combination site；It is based on
The secondary structure of aptamer Bor-B02 prediction, truncates aptamers Bor-B02.
9. a kind of try with the aptamer of borax specific binding in identification borax as described in claims 1 to 3 any one
Application in agent box.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910371702.3A CN110423754A (en) | 2019-05-06 | 2019-05-06 | Aptamer and utilization SELEX screening technique and application with borax specific binding |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910371702.3A CN110423754A (en) | 2019-05-06 | 2019-05-06 | Aptamer and utilization SELEX screening technique and application with borax specific binding |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110423754A true CN110423754A (en) | 2019-11-08 |
Family
ID=68407498
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910371702.3A Pending CN110423754A (en) | 2019-05-06 | 2019-05-06 | Aptamer and utilization SELEX screening technique and application with borax specific binding |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110423754A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113789332A (en) * | 2021-10-09 | 2021-12-14 | 昆明理工大学 | Aptamer specifically bound with p-hydroxybenzyl bisulfite and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107505463A (en) * | 2017-07-17 | 2017-12-22 | 昆明理工大学 | With the aptamer Sf B09 of sodium formaldehyde sulfoxylate specific bond and its application |
| CN108486119A (en) * | 2018-03-05 | 2018-09-04 | 昆明理工大学 | A kind of aptamer RhB-F02 and its application with rhodamine B specific binding |
-
2019
- 2019-05-06 CN CN201910371702.3A patent/CN110423754A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107505463A (en) * | 2017-07-17 | 2017-12-22 | 昆明理工大学 | With the aptamer Sf B09 of sodium formaldehyde sulfoxylate specific bond and its application |
| CN108486119A (en) * | 2018-03-05 | 2018-09-04 | 昆明理工大学 | A kind of aptamer RhB-F02 and its application with rhodamine B specific binding |
Non-Patent Citations (1)
| Title |
|---|
| 赵龙等: "食品安全快速检测技术的应用和不足", 《现代食品》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113789332A (en) * | 2021-10-09 | 2021-12-14 | 昆明理工大学 | Aptamer specifically bound with p-hydroxybenzyl bisulfite and application |
| CN113789332B (en) * | 2021-10-09 | 2023-08-18 | 昆明理工大学 | Nucleic acid aptamer specifically combined with p-hydroxybenzylsulfuronate and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bruno et al. | A novel screening method for competitive FRET-aptamers applied to E. coli assay development | |
| JP2674957B2 (en) | Electrochemiluminescence assay | |
| CN107614700A (en) | Genotype and phenotype coupling | |
| CN103513027B (en) | Establishment of novel ultrasensitiveness ELISA method | |
| WO2004009848A1 (en) | Microparticle based signal amplification for the detection of analytes | |
| KR101460450B1 (en) | DNA aptamer specifically binding to cadmium and uses thereof | |
| CN107462714B (en) | With the aptamer Sf-A09 of sodium formaldehyde sulfoxylate specific bond and its application | |
| CN107505463B (en) | With the aptamer Sf-B09 of sodium formaldehyde sulfoxylate specific bond and its application | |
| CN103725686B (en) | AFB 1aptamer AFB 1-20 and application | |
| US20240060969A1 (en) | Method of determining the presence or absence of a target analyte comprising using a reporter polynucleotide and a transmembrane pore | |
| Shin et al. | Recent advances in engineering aptamer-based sensing and recovery of heavy metals and rare earth elements for environmental sustainability | |
| CN110423754A (en) | Aptamer and utilization SELEX screening technique and application with borax specific binding | |
| CN105039346B (en) | A kind of aptamer and application with melamine specific bond | |
| CN110066804A (en) | A kind of aptamer with borax specific binding | |
| CN108251429A (en) | A kind of aptamer Met-G02 and its application with acephatemet specific binding | |
| US8981075B2 (en) | PSA binding aptamer and method for diagnosis of prostate cancer | |
| Hirose et al. | Strong and Specific Recognition of CAG/CTG Repeat DNA (5’‐dWGCWGCW‐3’) by a Cyclic Pyrrole‐Imidazole Polyamide | |
| CN107505464B (en) | With the aptamer Sf-E07 of sodium formaldehyde sulfoxylate specific bond and its application | |
| CN105177009B (en) | A kind of aptamer and application with α-amanita hemolysin specific bond | |
| CN105177011B (en) | A kind of aptamer and application with clenbuterol hydrochloride specific bond | |
| CN108486119B (en) | Aptamer RhB-F02 specifically bound with rhodamine B and application thereof | |
| CN104878015B (en) | A kind of bacterial endotoxin aptamer and its application | |
| CN104862314B (en) | A kind of and the aptamer of α amanita hemolysin specific bonds and application | |
| CN110286216A (en) | A kind of hemolytic relative gene O157:H7 detection method of quick visualization | |
| CN105039347B (en) | A kind of aptamer and application with glyphosate specific bond |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191108 |
|
| RJ01 | Rejection of invention patent application after publication |