CN109096391A - A kind of preparation method and application for the bionic Nano particles of silicon dioxide that polypeptide mediates - Google Patents
A kind of preparation method and application for the bionic Nano particles of silicon dioxide that polypeptide mediates Download PDFInfo
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- CN109096391A CN109096391A CN201810801924.XA CN201810801924A CN109096391A CN 109096391 A CN109096391 A CN 109096391A CN 201810801924 A CN201810801924 A CN 201810801924A CN 109096391 A CN109096391 A CN 109096391A
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- Prior art keywords
- silicon dioxide
- solution
- nano particles
- preparation
- silicon
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 title claims abstract description 242
- 239000000377 silicon dioxide Substances 0.000 title claims abstract description 123
- 235000012239 silicon dioxide Nutrition 0.000 title claims abstract description 67
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 37
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 37
- 239000011664 nicotinic acid Substances 0.000 title claims abstract description 34
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 33
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 74
- 230000008569 process Effects 0.000 claims abstract description 59
- 229910052710 silicon Inorganic materials 0.000 claims abstract description 53
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims abstract description 50
- 238000004153 renaturation Methods 0.000 claims abstract description 49
- 239000010703 silicon Substances 0.000 claims abstract description 48
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 47
- 239000010439 graphite Substances 0.000 claims abstract description 47
- 229910002804 graphite Inorganic materials 0.000 claims abstract description 47
- 230000001376 precipitating effect Effects 0.000 claims abstract description 31
- 108010014258 Elastin Proteins 0.000 claims abstract description 25
- 102000016942 Elastin Human genes 0.000 claims abstract description 24
- 229920002549 elastin Polymers 0.000 claims abstract description 24
- 239000008187 granular material Substances 0.000 claims abstract description 24
- 238000002156 mixing Methods 0.000 claims abstract description 19
- 108090000790 Enzymes Proteins 0.000 claims abstract description 10
- 102000004190 Enzymes Human genes 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 100
- 210000001783 ELP Anatomy 0.000 claims description 51
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 24
- 238000000746 purification Methods 0.000 claims description 21
- 238000001556 precipitation Methods 0.000 claims description 19
- 239000007787 solid Substances 0.000 claims description 15
- 238000005119 centrifugation Methods 0.000 claims description 14
- 239000007853 buffer solution Substances 0.000 claims description 13
- 230000015572 biosynthetic process Effects 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 9
- 230000002776 aggregation Effects 0.000 claims description 8
- 229910052681 coesite Inorganic materials 0.000 claims description 8
- 229910052906 cristobalite Inorganic materials 0.000 claims description 8
- 239000002243 precursor Substances 0.000 claims description 8
- 229910052682 stishovite Inorganic materials 0.000 claims description 8
- 229910052905 tridymite Inorganic materials 0.000 claims description 8
- 238000000137 annealing Methods 0.000 claims description 7
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims description 6
- 230000018044 dehydration Effects 0.000 claims description 6
- 238000006297 dehydration reaction Methods 0.000 claims description 6
- 230000003252 repetitive effect Effects 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 5
- 238000004220 aggregation Methods 0.000 claims description 5
- 210000003000 inclusion body Anatomy 0.000 claims description 5
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 5
- 238000002604 ultrasonography Methods 0.000 claims description 5
- 235000014393 valine Nutrition 0.000 claims description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 4
- 239000004472 Lysine Substances 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- CZDYPVPMEAXLPK-UHFFFAOYSA-N tetramethylsilane Chemical group C[Si](C)(C)C CZDYPVPMEAXLPK-UHFFFAOYSA-N 0.000 claims description 4
- 150000003680 valines Chemical class 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 3
- 238000013019 agitation Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000000703 high-speed centrifugation Methods 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 230000004845 protein aggregation Effects 0.000 claims description 3
- 230000012846 protein folding Effects 0.000 claims description 3
- 239000004408 titanium dioxide Substances 0.000 claims description 3
- XZWYZXLIPXDOLR-UHFFFAOYSA-N metformin Chemical compound CN(C)C(=N)NC(N)=N XZWYZXLIPXDOLR-UHFFFAOYSA-N 0.000 claims description 2
- VCZQFJFZMMALHB-UHFFFAOYSA-N tetraethylsilane Chemical compound CC[Si](CC)(CC)CC VCZQFJFZMMALHB-UHFFFAOYSA-N 0.000 claims description 2
- 230000007704 transition Effects 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 19
- 239000002245 particle Substances 0.000 abstract description 11
- 230000001965 increasing effect Effects 0.000 abstract description 4
- 239000003937 drug carrier Substances 0.000 abstract description 3
- 230000001404 mediated effect Effects 0.000 abstract description 3
- 238000009826 distribution Methods 0.000 abstract description 2
- 239000001963 growth medium Substances 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 16
- 239000002953 phosphate buffered saline Substances 0.000 description 14
- 239000007788 liquid Substances 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 238000004458 analytical method Methods 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 235000018102 proteins Nutrition 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 230000033558 biomineral tissue development Effects 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 239000003792 electrolyte Substances 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 6
- 239000013049 sediment Substances 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- KLOHDWPABZXLGI-YWUHCJSESA-M ampicillin sodium Chemical compound [Na+].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 KLOHDWPABZXLGI-YWUHCJSESA-M 0.000 description 5
- 229960001931 ampicillin sodium Drugs 0.000 description 5
- 238000006555 catalytic reaction Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000035484 reaction time Effects 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 235000013339 cereals Nutrition 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 4
- 230000005518 electrochemistry Effects 0.000 description 4
- 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 4
- 229920002521 macromolecule Polymers 0.000 description 4
- 125000004430 oxygen atom Chemical group O* 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000005054 agglomeration Methods 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 description 3
- 229910010272 inorganic material Inorganic materials 0.000 description 3
- 239000011147 inorganic material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 230000036632 reaction speed Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- LIVNPJMFVYWSIS-UHFFFAOYSA-N silicon monoxide Chemical compound [Si-]#[O+] LIVNPJMFVYWSIS-UHFFFAOYSA-N 0.000 description 3
- 229910052814 silicon oxide Inorganic materials 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 229910001868 water Inorganic materials 0.000 description 3
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000011953 bioanalysis Methods 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 150000001721 carbon Chemical group 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 125000002091 cationic group Chemical group 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 229910001431 copper ion Inorganic materials 0.000 description 2
- 239000000287 crude extract Substances 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000008151 electrolyte solution Substances 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000002086 nanomaterial Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- VDGJOQCBCPGFFD-UHFFFAOYSA-N oxygen(2-) silicon(4+) titanium(4+) Chemical compound [Si+4].[O-2].[O-2].[Ti+4] VDGJOQCBCPGFFD-UHFFFAOYSA-N 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000007981 phosphate-citrate buffer Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 229920000768 polyamine Polymers 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000011856 silicon-based particle Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- 241000206761 Bacillariophyta Species 0.000 description 1
- 238000007445 Chromatographic isolation Methods 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 241000790917 Dioxys <bee> Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229910018557 Si O Inorganic materials 0.000 description 1
- 229910003978 SiClx Inorganic materials 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000005267 amalgamation Methods 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000000035 biogenic effect Effects 0.000 description 1
- 230000003592 biomimetic effect Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 235000019628 coolness Nutrition 0.000 description 1
- -1 copper ion Sodium alkyl sulfate Chemical class 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000003208 gene overexpression Methods 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229910003471 inorganic composite material Inorganic materials 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 239000013618 particulate matter Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010850 salt effect Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B33/00—Silicon; Compounds thereof
- C01B33/113—Silicon oxides; Hydrates thereof
- C01B33/12—Silica; Hydrates thereof, e.g. lepidoic silicic acid
- C01B33/18—Preparation of finely divided silica neither in sol nor in gel form; After-treatment thereof
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nanotechnology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Composite Materials (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- General Physics & Mathematics (AREA)
- Materials Engineering (AREA)
- Crystallography & Structural Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
A kind of preparation method and application for the bionic Nano particles of silicon dioxide that polypeptide mediates, realizes quick obtaining class elastin polypeptide ELP40 by using improved plan (increasing the renaturation process to precipitating)；Class elastin polypeptide ELP40 is mediated in silicon dioxide granule precipitating, improving to silicon source solution makes itself and discrete graphite solution ultrasonic mixing, being sufficiently mixed for particle can be achieved, and then it can get the bionic Nano particles of silicon dioxide of the spherical structure of rule (and particle diameter distribution more concentrated, particle diameter size is in 70nm between 400nm), reaction condition is mild, equipment is simple and convenient to operate, is easy to reach industrially scalable, has important industrial application value and huge application potential.Industrially, it can be applied to immobilised enzymes field；Biomedical aspect can be used as good pharmaceutical carrier, especially slow releasing pharmaceutical.
Description
Technical field
It is specifically a kind of bionical the present invention relates to the inorganic material of the artificial synthesized particular nanostructure of method by biology
The preparation of Nano particles of silicon dioxide is based especially on the preparation method of the bionic Nano particles of silicon dioxide of polypeptide mediation.
Background technique
There is a kind of very universal phenomenons in nature --- biomineralization.In biomineralization phenomenon, titanium dioxide
Silicon accounts for larger a part, is widely present in such as sponge, bacterium, unicellular alga, higher plant and protozoan.
Scientist has found through in-depth study, compared to silica caused by artificial synthesized technology, these bio-silicon dioxides tool
The specific function and complicated structure for thering is the former to be difficult to realize.In addition to this, this kind of biomineralization process belongs to very typical
Green low power consuming synthetic technology do not need high temperature, the high pressure of conventional synthesis technology because it is formed under relatively mild conditions
The more harsh synthesis condition with extreme ph values etc..
Another important feature of biomineralization is: organic matter can be with self assembly organic polymer or using biology greatly
Molecular template synthesizes the organic and inorganic composite material such as the fine structure of Nano grade, and scientist can follow this feature to close
At with the vdiverse in function and compound inorganic material of form.Therefore, such research direction also accounts in materials chemistry research field
It is template or simulation biomolecule guidance inorganic material (such as two to synthesize macromolecule organic according to important a part
Silica) to new construction manufacture organic molecule coating or nano materials, this kind of mode in terms of have
Excellent application prospect.
During biomineralization, (organism contains some with specific function or the protein of structure etc. large biological molecule
Organic macromolecule) phase between some functional organic macromolecules and inorganic matter ion can occur for the interfaces of functional groups
Why interaction, i.e. biogenic mineral have special assembling mode and abnormal fine structure, are because of such functional macromolecule
The structure of inorganic mineral and form are regulated and controled from nanoscale by the functional group of itself, that is to say, that albumen
The organic substances such as matter play considerable regulating and controlling effect during biomineralization.
By the silicon mineralization process of ocean eucaryote Diatomeae in nature and cavernous body, used in the prior art from biology
The amine molecule or macromolecular, polypeptide and protein of bioactive molecule (such as silaffins), synthesis that body extracts, synthesizing bionic
Silica, such as application No. is functional bionic Nano particles of silicon dioxide of 200610124462.X and preparation method thereof,
It uses cationic polyamine microgel as functional template, makes silica bionical deposition in situ at ambient conditions, obtains
Cationic polyamine/SiO2 hydridization functional nanoparticles.Wherein, the functional template used can not be used repeatedly, because
The preparation process of this Nano particles of silicon dioxide is complex, increases its cost of implementation.
For this purpose, the prior art has investigated a type elastin polypeptide (ELPs) to substitute existing function on the basis of this
Property template, such elastin polypeptide is a kind of to be connected by Val-Pro-Gly-Xaa-Gly (VPGXG) pentapeptide repetitive unit sequence
The artificial polymer of composition, wherein Xaa can be any amino acid in addition to proline (Pro), mostly lysine, valine,
Phenylalanine.It is mentioned in the document that such elastin polypeptide (ELPs) is delivered disclosed in the following inventor: [1] Huang Kai
Ancestor, Jingjing LI, Li Wei, Ge Huihua, Wang Wenyan, from the beginning design, non-chromatogram purification and the salt effect of Zhang Guangya class elastin polypeptide
Answer [J] bioengineering journal, 2011,27 (4): 653-658. [2] Fu Xiaoping, Wang Wenyan, Zhang Guangya is more with class elastin laminin
The expression plasmid building and its non-chromatogram purification [J] microorganism journal for zytase that peptide is label, 2012,52 (1):
90-95. [3] Li Cuncun, Zhang Guangya enzyme oriented immobilization method and progress [J] the chemical industry of application progress, 2013,32
(10): one kind disclosed in the patent of invention application No. is 201010562100.5 of 2467-2474. [4] inventor's earlier application
The fusion tag albumen and its encoding gene and preparation method of non-chromatographic isolation can be carried out to target protein.
Summary of the invention
The brief overview about the embodiment of the present invention is given below, in order to provide about certain aspects of the invention
Basic comprehension.It should be appreciated that outlined below is not about exhaustive general introduction of the invention.It is not intended to determine this hair
Bright key or pith, nor is it intended to limit the scope of the present invention.Its purpose only provides certain in simplified form
A little concepts, taking this as a prelude to a more detailed description discussed later.
According to the one aspect of the application, a kind of preparation side of bionic Nano particles of silicon dioxide that polypeptide mediates is provided
Method comprising:
Step 1: using the class elastin polypeptide ELPs of ITC method of purification preparation high-purity；
In this step, the ITC method of purification of the prior art is usually following process: being allowed to occur to the heating of ELPs solution first
Phase transformation, centrifugation is gone to collect and be precipitated under the conditions of being higher than Tt；Then, precipitated with the buffer solution of pre-cooling, under cryogenic from
The heart collects supernatant, this is the ELPs purified；Above-mentioned ITC purification process is repeated, until ELPs after purification reaches purity；Its
In, number needed for ITC purification process generally goes through 2~5 ITC purifying because the difference of destination protein and ELPs can be variant
Process can be obtained the albumen of higher degree；
The present invention realizes the class elastin polypeptide ELPs of quickly preparation high-purity using improved ITC method of purification.Its
Principle is, due to energy transmission and local heat production etc., is difficult to the broken of large volume cell suspension, part is not broken in this way
Chopping fine born of the same parents mix with inclusion body, bring difficulty to later-period purification, to reduce the inclusion body in the precipitating being collected after centrifugation, this
Apply carrying out renaturation process to the precipitating after centrifugation, the ratio of inactive solid particle is reduced, to improve purifying speed.Its
In, renaturation process is carried out to the precipitating after centrifugation and is specifically included: precipitating being carried out fastly for (about -18 DEG C to 0 DEG C) at low temperature first
Fast (in 10 seconds) dehydration, and appropriate shatter (can be shatter with ultrasound) be carried out to the precipitating after dehydration, obtain solid-state
Then grain object is sieved to the particulate solid, selection by winnowing or high-pressure electrostatic absorption (using denaturation salt crystallization specific gravity), will be big
The removal of part inclusion body, the precipitating after obtaining renaturation.Above-mentioned dehydration process is needed quick and of short duration and is carried out at low temperature,
It can avoid influencing the stability and activity of the albumen after renaturation.Renaturation process of the above process as first time, it is preferred multiple
Property process further includes secondary renaturation process: by the particulate solid after the renaturation process of first time at (about -18 DEG C of low temperature
To -10 DEG C) under refolding to reduce the formation of protein aggregation, when the intermediate for forming aggregation is reduced, rapidly
Temperature is improved into about 10 degree or so (temperature after guaranteeing raising temperature is still less than 0 degree), to promote protein folding renaturation, thus
It can get the solid granulates of high annealing efficiency.In special circumstances, above-mentioned second of renaturation process can execute 2 times it is (general primary
) to further increase annealing efficiency.Meanwhile secondary renaturation process can also be the side for carrying out renaturation with renaturation solution
Case, such as particulate solid is slowly added to be mixed with renaturation solution (the general buffer equilibrium liquid, such as 12 of copper ion
Sodium alkyl sulfate solution, phosphate buffered saline solution PBS etc.), preset time (example is reacted under specific temperature (such as 25-35 DEG C)
Such as 1 hour), the terminations such as inhibitor, renaturation agent reaction is added, centrifugation finally is carried out to the solution and is precipitated.It is, in general, that
The annealing efficiency of protein is 20% or so, and the annealing efficiency of the application is up to 60%.
The application increases the renaturation process process to precipitating on the basis of existing technology, substantially increases purifying speed
Degree uses it is demonstrated experimentally that the scheme of the prior art needs to obtain the albumen of higher degree by 2~5 ITC purification process
The scheme of the application, it is only necessary to which 1 time is that can reach required purity.Moreover, product is solid-state after above-mentioned renaturation process
Grain object (partial size than original sediment is smaller) has dispersibility well, is more advantageous to it in point of step 2 mixed solution
It dissipates and solution comes into full contact with reaction, accelerate reaction speed.In addition, increasing the scheme phase of the scheme and the prior art of renaturation process
Than obtaining extraordinary facilitation in the preparation process of subsequent silica since its activated protein becomes more.
Step 2: class elastin polypeptide ELPs mediates silicon dioxide granule precipitating, obtains bionical silica dioxide nano particle
Son；Specifically include following process: the class elastin polypeptide ELPs that step 1 obtains is dissolved in PBS buffer solution, and to obtain ELPs molten
Liquid, the concentration range of class elastin polypeptide ELPs are 10 μm of ol/L-100 μm of ol/L, pH=(6.9-7.1)；By SiO2Precursor
It is dissolved in preparation silicon source solution, SiO in solution2The optional tetramethylsilane of precursor or tetraethyl silane；The ELPs for being a by volume
Solution is mixed with the silicon source solution that volume is b, and a: b=(9-11): 1；Stand preset time at room temperature after mixing
(5min), high speed centrifugation (10,000rpm, be centrifuged 3min), collects precipitation of silica, it is multiple that silica/ELPs can be obtained
Condensation material (bionic Nano particles of silicon dioxide).
The bionic Nano particles of silicon dioxide that existing polypeptide mediates silicon dioxide granule precipitating to obtain is hardly formed rule
Spherical shape, this is because SiO2It is a kind of space multistory network structure, four oxygen atoms of each silicon atom and surrounding are formed
One Si, O positive tetrahedron, i.e. silicon atom are located at the center of positive tetrahedron, and four oxygen atoms are located at four vertex of positive tetrahedron
On, each oxygen atom participates in forming 2 Si, O positive tetrahedrons, i.e. four oxygen atoms of each silicon atom and surrounding can be with shape
At four Si-O singly-bounds, and in microstructure, it is easy to that agglomeration occurs in silicon source solution, is easy to produce aggregation,
It so when silicon source solution is mixed with ELPs solution, is unable to fully mix, therefore the Nano particles of silicon dioxide finally obtained is normal
It is subjected to interference and regular spherical shape can not be obtained.Therefore, the application equally carries out to improve the above problem for this step
It improves, silicon source solution is improved, development is as follows: preparing silicon source solution: the SiO for be c (unit for gram) by quality2
Precursor (such as tetramethylsilane TMOS) is dissolved in the hydrochloric acid that concentration is d (unit mmol/L), constant volume v (unit mL)
Obtain silicon source solution；Prepare discrete graphite solution: it is that (unit is d that the graphite that quality is e (unit be gram), which is dissolved in concentration,
Mmol/L in hydrochloric acid), wherein c > 5e, constant volume v (unit mL) then carry out ultrasound and the discrete graphite solution of acquisition are blended；
Then silicon source solution and discrete graphite solution are mixed into simultaneously high-speed stirred preset time, mixing silicon source solution can be obtained；Wherein
Mixing silicon source solution is preferably obtained by following process: hydrochloric acid is added in graphite, carrying out ultrasonic agitation preset time, (at least 3 is small
When), suspension is obtained, then by the quickly mixing (in several seconds) of suspension and silicon source solution, when being stirred default for 35-60 DEG C
Between (to be generally more than 30min), can be obtained mixing silicon source solution.
Wherein, the space structure of graphite is a kind of layer structure, and in each layer, each carbon atom participates in forming 3
Planar regular hexagonal, by by the layer structure of graphite by SiO2Space network carry out it is decentralized, greatly reduce it
Agglomeration, therefore when silicon source solution is mixed with ELPs solution, it can be achieved that be sufficiently mixed, and then can get the spherical junctions of rule
The bionic Nano particles of silicon dioxide of structure.Meanwhile by the size of scanning electron microscope and transmission electron microscope observing silicon dioxide granule and
Pattern obtains silicon dioxide granule in the spherical shape of rule, and particle diameter size concentrates on 70nm between 400nm.
In addition, mixing silicon source solution is due to joined graphite, ELPs solution with mix two obtained after silicon source solution mixes
Have graphite inside silicon oxide precipitation, for this purpose, according to graphite and silica is non-conductive, can be by preparing electrolyte and be powered
(it is inserted into two electrodes in the electrolytic solution, when adding certain voltage between two electrodes with power supply, graphite is adsorbed to electrode, so
The final sediment of electrolyte is bionic Nano particles of silicon dioxide afterwards), the preparation and galvanization sheet of above-mentioned electrolyte
The technical staff in field sees associated materials acquisition, such as the logical work in lattice Lars, Jia Lide etc. are translated: " electrochemistry outline ", science
Publishing house, Beijing, 1958.(S.Glasstone, An lntroduction to Electrochemistry, Van
Nostrand, New York, 1947.), repeats no more herein.
In addition, being not the more high titanium dioxide finally obtained of quality of graphite in discrete graphite solution under experimental conditions
The particle of silicon particle is more, form is better, the mass percent concentration ω of graphite, ω=quality e*10-3(graphite rubs by/12*v
Your quality is 12g/mol, discrete graphite liquor capacity mass concentration=(molal weight × mmol/1000 of graphite)/l=g/
L), by being fitted to experimental data, the mass percent concentration ω for obtaining graphite is preferably 3wt%-3.7wt%, can also
It is expressed as 5.5e < c < 7e.
Preferably, the amino acid sequence of the class elastin polypeptide ELPs of above-mentioned generation is ELP [KV8F-s], ELP [KV8F-
S] refer in five peptide unit of continuous 10 VPGXG, the 4th Xaa of each pentapeptide has 1 lysine (K), 8 valines
(V), 1 phenylalanine (F), s, which refers to, has s a using VPGXG as the repetitive sequence of unit.Class elastin polypeptide ELPs is a kind of
Artificial gene's engineered protein of energy response temperature change, it includes several repetitive sequences (VPGXG) n, and wherein X, which is represented, removes proline
Any amino acid in addition, the design combination of different X can generate the ELPs of different properties.According to the experimental verification of the application and
The mediation of the application, and the variation model of repeat number s also may be implemented in inference, other ELPs sequences for having similar amino acid
It encloses for 10---300.Such as s=40, then ELPs is denoted as ELP40, and gene order is then ELP [KV8F-40], ELP [KV8F-40]
Refer in five peptide unit of continuous 10 VPGXG, the 4th Xaa of each pentapeptide has 1 lysine (K), 8 valines (V), 1
A phenylalanine (F), 40 i.e. refer to have 40 using VPGXG as the repetitive sequence of unit.
According to the another aspect of the application, a kind of answering by bionic Nano particles of silicon dioxide obtained by the above method is provided
With, wherein the bionic Nano particles of silicon dioxide is used for the self-retaining of enzyme.
According to the application's in another aspect, providing a kind of answering by bionic Nano particles of silicon dioxide obtained by the above method
With the carrier for the slow releasing pharmaceutical that the bionic Nano particles of silicon dioxide is used as.
The application through the above scheme, compared with prior art, has the advantage that
1, class elastin polypeptide ELPs using ELP40 (inventor's earlier application application No. is 201510042517.1
Patent of invention disclosed in a kind of preparation method of protein molecular weight standard its gene order is disclosed) realize, ELP40 is anti-
Answer the front and back ELP- silicon dioxide hybrid materials that can retain the property of its phase transformation, therefore finally obtain than traditional chemistry or
The silica of bioanalysis synthesis has more extensive application prospect.In addition, by experimentation it is found that ELP40 is both catalysis two
The catalyst of the formation of silica itself is embedded again and is fixed on the silica supports that oneself catalysis is formed, the reaction
Process is both the process of the process that silica is formed and ELP40 self-catalysis immobilization, and the reaction condition is mild, equipment is simple
It is single, easy to operate, be easy to reach industrially scalable, have important industrial application value and huge application potential；Industrially,
It can be applied to immobilised enzymes field；Biomedical aspect can be used as good pharmaceutical carrier, especially slow releasing pharmaceutical, tool
There is extraordinary application prospect.
2, in ITC purification process, the renaturation process to precipitating is increased, substantially increases purifying speed, the prior art
Scheme need to obtain the albumen of higher degree by 2~5 ITC purification process, it is demonstrated experimentally that using the side of the application
Case, it is only necessary to which 1 time is that can reach required purity；Moreover, product is particulate solid (than original after above-mentioned renaturation process
Sediment partial size it is smaller), have well dispersibility, be more advantageous to it and filled in the dispersion of step 2 mixed solution and solution
Divide haptoreaction, accelerates reaction speed.In addition, increase renaturation process scheme compared with the scheme of the prior art, due to its work
Property albumen become it is more, extraordinary facilitation is obtained in the preparation process of subsequent silica；
3, class elastin polypeptide ELPs is mediated in silicon dioxide granule precipitating, improving to silicon source solution makes it
With discrete graphite solution ultrasonic mixing, the space network of silica is prevented by the layer structure of graphite in microstructure
Mutual aggregation, so that the spacing of silicon dioxide granule is become larger, and it piles up more loose, this loose structure can make
SiO2Other molecules of precursor and ELPs solution come into full contact with, and to form loose reticular structure, therefore work as improved silicon source
, it can be achieved that being sufficiently mixed when solution is mixed with ELPs solution, and then the bionical silica that can get the spherical structure of rule is received
Rice corpuscles (and particle diameter distribution is more concentrated, particle diameter size is in 70nm between 400nm, and the partial size of the prior art is generally several
Between ten nanometers to tens microns)；
4, in addition, during improving to silicon source solution, also by being fitted to experimental data, graphite is obtained
Mass percent concentration ω is preferably 3wt%-3.7wt%, can also be expressed as 5.5e < c < 7e, obtains in such cases more preferably
Bionic Nano particles of silicon dioxide, shape is more regular, and partial size is more concentrated.
Detailed description of the invention
The present invention can be by reference to being better understood below in association with description given by attached drawing.The attached drawing
Together with following detailed description comprising in the present specification and forming a part of this specification, and it is used to further
The preferred embodiment and explanation the principle of the present invention and advantage illustrated the present invention.In the accompanying drawings:
Fig. 1 is that ELP40 mediates the silicon dioxide granule sem analysis to be formed；
Fig. 2 a is that ELP40 mediates the silicon dioxide granule tem analysis schematic diagram one to be formed；
Fig. 2 b is that ELP40 mediates the silicon dioxide granule tem analysis schematic diagram two to be formed；
Fig. 3 is influence of the ELP40 concentration to precipitation of silica amount；
Fig. 4 is influence of the solution ph to precipitation of silica amount；
Fig. 5 is influence of the reaction temperature to precipitation of silica amount；
Fig. 6 is influence of the reaction time to precipitation of silica amount；
Fig. 7 is the ELP40SDS-PAGE analysis discharged in silicon dioxide granule；
Fig. 8 is the SDS-PAGE analysis that albumen ELP40 is expressed and purified.
Specific embodiment
Illustrate the embodiment of the present invention below with reference to accompanying drawings.It should be noted that for purposes of clarity, attached drawing and explanation
In the expression and description of component unrelated to the invention, known to persons of ordinary skill in the art and processing is omitted.
The present invention provides a kind of fast preparation method of bionic Nano particles of silicon dioxide that polypeptide mediates comprising:
Step 1: using the class elastin polypeptide ELP40 of ITC method of purification preparation high-purity；It is specific real as one
Example, the process specifically include:
Preparation: cleaning superclean bench, ultraviolet sterilization 20min；Ampicillin sodium presses 1: 1000 (ampicillin
Sodium: culture medium, v/v) ratio, as the culture medium of 10mL adds 10 μ L；Glycerol stock is by 1: 100 (strain: culture medium, v/v) ratio
Inoculum concentration is added in LB liquid medium, as the culture medium of 10mL adds the strain of 100 μ L；Culture medium is placed in 37 DEG C, 200rpm
Constant-temperature table culture 10-12h.
Expand culture and inducing expression: before inoculation, clearing up superclean bench, and ultraviolet sterilization 20min；Inoculation: ammonia benzyl is green
Mycin sodium is added in TB culture medium in 1: 1000 ratio, the strain of activation in 1: 100 ratio, such as the culture medium of 200mL
Add the ampicillin sodium of 200 μ L and the strain of 2mL；Expand culture: will be inoculated with that culture medium is placed in 37 DEG C, 200rpm constant temperature shakes
Bed culture 2-4h, OD600Value is between 0.6-0.8, at this point, thallus is in logarithmic growth phase；
Inducing expression: IPTG is added in above-mentioned culture medium the (training of such as 200mL by 1: 200 (IPTG: culture medium, v/v)
Support base add 1mL), by culture medium be placed in 37 DEG C, 200rpm constant-temperature table continue culture for 24 hours induce target gene overexpression.
Cell is collected and is crushed: above-mentioned cultured bacterium solution being sub-packed in 500mL centrifuge tube and (does not exceed centrifugation tube body
Long-pending 2/3), 4000rpm, room temperature is centrifuged 20min；Supernatant is discarded, by the ratio of 1: 25 (PBS buffer solution: bacterium solution, v/v)
PBS buffer solution is added, twice, 4 DEG C, 10000rpm is centrifuged 10min to washing thalline, collects cell；Supernatant is discarded, by 1: 25
The ratio of (PBS buffer solution: bacterium solution, v/v) is added PBS buffer solution, resuspended bacterium solution, as carrying out ultrasonication in ice bath.
Ultrasonic Pulverization condition: power 300W, ultrasonic 2s are spaced 4s, and cycle-index is 200 times, ultrasonic time 12min.Clasmatosis
Liquid is centrifuged 10min, supernatant is cell crude extract in 4 DEG C, 10000rpm.
The ITC of albumen is purified: solid NaCl (2mol/L) is added into cell crude extract, triggering ELPs is undergone phase transition, and 37
After DEG C water bath with thermostatic control 20min, 30 DEG C, 10000rpm, it is centrifuged 10min；Abandon supernatant, collect precipitating, to the precipitating after centrifugation into
Row renaturation process；The PBS buffer solution of 4 DEG C of pre-coolings is added in sediment after renaturation process to dissolve the albumen of precipitating, first
Precipitating piping and druming is got up with liquid-transfering gun, is mixed well, 4 DEG C of ice bath 30min (dissolving the target protein of precipitating sufficiently),
10000rpm is centrifuged 10min, collects supernatant；Final supernatant is pure ELP40.The SDS-PAGE of albumen ELP40 expression and purifying
Analysis is referring to Fig. 8.
Above-mentioned class elastin polypeptide ELPs is realized using ELP40 in the application, and the gene order of ELP40 is ELP
[KV8F-40], ELP [KV8F-40] refer in five peptide unit of continuous 10 VPGXG, the 4th Xaa of each pentapeptide has 1 to rely
Propylhomoserin (K), 8 valines (V), 1 phenylalanine (F), 40 i.e. refer to have 40 using VPGXG as the repetitive sequence of unit.The sequence
Column have carried out strain Patent Deposit (China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC
NO.4185, preservation date: on September 17th, 2010), and disclosed in the patent of invention application No. is 201010562100.5.
In this step, the ITC method of purification of the prior art is usually following process: being allowed to occur to the heating of ELPs solution first
Phase transformation, centrifugation is gone to collect and be precipitated under the conditions of being higher than Tt；Then, precipitated with the buffer solution of pre-cooling, under cryogenic from
The heart collects supernatant, this is the ELPs purified；Above-mentioned ITC purification process is repeated, until ELPs after purification reaches purity；Its
In, number needed for ITC purification process generally goes through 2~5 ITC purifying because the difference of destination protein and ELPs can be variant
Process can be obtained the albumen of higher degree.The application increases the step that renaturation process is carried out to precipitating in the prior art
Suddenly, the multiple purification process of the prior art is shorten to 1 time.
Wherein, renaturation process is carried out to the precipitating after centrifugation to specifically include: being deposited in -12 DEG C of temperature after making centrifugation first
Under carry out fast dewatering processing in 10 seconds, and it is shatter (can with ultrasound shatter) to carry out appropriateness to the precipitating after dehydration, obtains
Particulate solid is obtained, is then sieved to the particulate solid, the absorption of selection by winnowing or high-pressure electrostatic (utilizes denaturation salt crystallization ratio
Weight), most of inclusion body is removed, the precipitating after obtaining renaturation.Above-mentioned dehydration process needs are quick and of short duration and low
Temperature is lower to be carried out, and can avoid the stability and activity that influence the albumen after renaturation.Renaturation process of the above process as first time,
Preferred renaturation process further includes secondary renaturation process: by the particulate solid after the renaturation process of first time at -12 DEG C
Refolding rapidly will be warm when the intermediate for forming aggregation is reduced with the formation for reducing protein aggregation under low temperature
Degree improves about 10 degree or so (about 2 degree), to promote protein folding renaturation, finally slowly restores room temperature, to can get high multiple
The solid granulates of property efficiency.In special circumstances, above-mentioned second of renaturation process can execute 2 times (general primary) with into one
Step improves annealing efficiency.Meanwhile secondary renaturation process can also be the scheme for carrying out renaturation with renaturation solution, such as by solid-state
Particulate matter is slowly added to be mixed with the renaturation solution of copper ion, and (general buffer equilibrium liquid, such as lauryl sodium sulfate are molten
Liquid, phosphate buffered saline solution PBS etc.), preset time (such as 1 hour) is reacted under specific temperature (such as 25-35 DEG C), then plus
Enter the terminations such as inhibitor, renaturation agent reaction, centrifugation finally is carried out to the solution and is precipitated.It is, in general, that the renaturation of protein
Efficiency is 20% or so, and the annealing efficiency of the application is up to 60%.
The application increases the renaturation process process to precipitating on the basis of existing technology, substantially increases purifying speed
Degree uses it is demonstrated experimentally that the scheme of the prior art needs to obtain the albumen of higher degree by 2~5 ITC purification process
The scheme of the application, it is only necessary to which 1 time is that can reach required purity.It is entire multiple although the application joined renaturation process
Property process be simply easily achieved, therefore, it is whole comparatively, the scheme of the application can realize faster it is highly purified.Very not
Only in this way, product is particulate solid (partial size than original sediment is smaller) after above-mentioned renaturation process, has and divide well
Property is dissipated, it is more advantageous in the dispersion of step 2 mixed solution and solution and comes into full contact with reaction, accelerate reaction speed.In addition, increasing
The scheme of renaturation process is compared with the scheme of the prior art, since its activated protein becomes more, in the preparation of subsequent silica
Extraordinary facilitation is obtained in the process.
Step 2: class elastin polypeptide ELP40 mediates silicon dioxide granule precipitating, obtains bionical silica dioxide nano particle
Son；Specifically include following process:
The class elastin polypeptide ELP40 that step 1 obtains is dissolved in PBS buffer solution and obtains ELP40 solution, concentration is
100 μm of ol/L, pH=7；
Silicon source solution is made in the TMOS for taking the 1mmol/L of 10 μ L to be dissolved in hydrochloric acid；Meanwhile the application also to silicon source solution into
Row improves, and specifically the preparation method is as follows: preparing discrete graphite solution: it is 1mmol/L that the graphite that quality is 0.2g, which is dissolved in concentration,
Hydrochloric acid in, be settled to 10mL, then carry out ultrasound and be blended to obtain discrete graphite solution；Then silicon source solution made of will be above-mentioned
With the mixing of discrete graphite solution and high-speed stirred 30 minutes, mixing silicon source solution can be obtained；Wherein mixing silicon source solution can lead to
It crosses following process to obtain: hydrochloric acid is added in graphite, ultrasonic agitation preset time (at least 3 hours) is carried out, obtains suspension, then
By the quickly mixing (in several seconds) of suspension and silicon source solution, preset time is stirred at 35-60 DEG C (generally will be more than
30min), mixing silicon source solution can be obtained；
ELP40 solution and mixing silicon source solution (improved silicon source solution) are mixed, it is quiet at room temperature after mixing
It sets preset time (5min), high speed centrifugation (10,000rpm, be centrifuged 3min) collects precipitation of silica；
Silica/ELP40 composite material, i.e. bionic Nano particles of silicon dioxide can be obtained.
In addition, mixing silicon source solution is due to joined graphite, ELPs solution with mix two obtained after silicon source solution mixes
Have graphite inside silicon oxide precipitation, for this purpose, according to graphite and silica is non-conductive, can be by preparing electrolyte and be powered
(it is inserted into two electrodes in the electrolytic solution, when adding certain voltage between two electrodes with power supply, graphite is adsorbed to electrode, so
The final sediment of electrolyte is bionic Nano particles of silicon dioxide afterwards), the preparation and galvanization sheet of above-mentioned electrolyte
The technical staff in field sees associated materials acquisition, such as the logical work in lattice Lars, Jia Lide etc. are translated: " electrochemistry outline ", science
Publishing house, Beijing, 1958.(S.Glasstone, An lntroduction to Electrochemistry, Van
Nostrand, New York, 1947.), repeats no more herein.
Wherein, the space structure of graphite is a kind of layer structure, and in each layer, each carbon atom participates in forming 3
Planar regular hexagonal, by by the layer structure of graphite by SiO2Space network carry out it is decentralized, greatly reduce it
Agglomeration, therefore when silicon source solution is mixed with ELPs solution, it can be achieved that be sufficiently mixed, and then can get the spherical junctions of rule
The bionic Nano particles of silicon dioxide of structure.The size of obtained bionic Nano particles of silicon dioxide is 700nm to 1000nm, bionical
The mass percent of silica is 10%-99% in Nano particles of silicon dioxide.Meanwhile passing through scanning electron microscope and transmission electron microscope
The size and pattern for observing silicon dioxide granule obtain silicon dioxide granule in the spherical shape of rule, and particle diameter size concentrates on
70nm is between 400nm.
In order to for scanning electron microscope and projection electron microscope analysis, then need to test finally obtained silica/
ELP40 composite material is placed in frozen drying 12h, then dry 12h in 70 DEG C of vacuum ovens.ELP40, which is mediated, to be formed
Silicon dioxide granule sem analysis is shown in Figure 1, and ELP40 mediates the silicon dioxide granule tem analysis to be formed a and figure referring to fig. 2
Shown in 2b, in Fig. 2 a, silicon dioxide granule model S4800 electron microscope, with operating voltage be 50,000 volts, working distance
From for 8.2mm, amplify 40,000 times of obtained tem analysis figures, Fig. 2 b is electron microscopic of the silicon dioxide granule in model S4800
Mirror amplifies 1.3 ten thousand times of obtained tem analysis figures with operating voltage for 50,000 volts, operating distance 8.5mm.
In addition, influence of the ELP40 concentration to precipitation of silica amount be referring to Fig. 3, when ELP40 concentration is 10 μm of ol/L-100
When between μm ol/L, precipitation of silica amount increases with the increase of ELP40 concentration.In this concentration range, reaction is still
Not up to saturation state.
Fig. 4 is influence schematic diagram of the solution ph to precipitation of silica amount, the reaction condition for being as seen from the figure 7.0 in pH
Under, the precipitation of silica amount of generation is most.It therefore, is that ELP40 mediates the most suitable anti-of silica mineralising when pH is 7.0
Answer pH value.
Fig. 5 is influence schematic diagram of the reaction temperature to precipitation of silica amount, when reaction temperature is 45 DEG C, generates dioxy
SiClx precipitation capacity is most.It therefore, is the optimal reactive temperature that ELP40 mediates silica mineralising when temperature is 45 DEG C.
Fig. 6 is influence of the reaction time to precipitation of silica amount, as seen from the figure, as the reaction time gradually increases, when
After reaction time reaches 10min, the precipitation of silica amount of generation tends towards stability.Therefore, when reacted between when being set as 10min,
Preferable reaction result can be obtained, while also can save the reaction time.
Fig. 7 is the ELP40 SDS-PAGE analysis discharged in silicon dioxide granule, and swimming lane 1 is to mediate silica heavy in figure
ELP40 before product, swimming lane 2 are the ELP40 for dissolving silica using sodium hydroxide and releasing, it can be seen that EL40P
Molecular weight mediate silica formed before and after there is no variation.In addition, ELP40 before the reaction after can retain its phase
The property of change, therefore, the silica that ELP40- silicon dioxide hybrid materials are synthesized than traditional chemistry or bioanalysis is perhaps
There is more extensive application prospect.From figure, still further it can be seen that EL40P is embedded in titanium dioxide silicon grain in the process of reaction simultaneously
Sub- the inside.Therefore, in this reaction process, the formation of silica is synchronous progress with the immobilization of ELP and completes.It is comprehensive
Upper described, ELP40 is both the catalyst for being catalyzed the formation of silica, itself is embedded again and is fixed on what oneself catalysis was formed
On silica supports.The reaction process is both the process of the process that silica is formed and ELP40 self-catalysis immobilization.
The reaction condition is mild, equipment is simple and convenient to operate, is easy to reach industrially scalable, with important industrial application value and
Huge application potential.Industrially, it can be applied to immobilised enzymes field；Biomedical aspect can be used as good drug
Carrier, especially slow releasing pharmaceutical.
In addition, being not the more high titanium dioxide finally obtained of quality of graphite in discrete graphite solution under experimental conditions
The particle of silicon particle is more, form is better, the mass percent concentration ω of graphite, ω=quality e*10-3/ 12*v, (graphite rubs
Your quality is 12g/mol, discrete graphite liquor capacity mass concentration=(molal weight × mmol/1000 of graphite)/l=g/
L), v is the volume of discrete graphite solution, and unit mL obtains the mass percent of graphite by being fitted to experimental data
Concentration ω is preferably 3wt%-3.7wt%, 5.5e < c < 7e, c SiO2The quality of precursor (TMOS).
The application tests the yield and grain that ELP40 biomimetic mineralization forms silica at different conditions through the above steps
The relationship of sub- size pattern, the results showed that it is 7.0 that ELP40, which mediates the optimal reaction pH of silica mineralising, optimal reactive temperature
Be 45 DEG C, precipitation of silica amount increases with the increase of ELP40 concentration, when reacted between reach two generated after 10min
Silicon oxide precipitation amount tends towards stability.It is the spherical shape of rule, diameter 70-400nm that ELP40, which mediates the silicon dioxide granule to be formed,.
The acquisition scheme of experimental material in the present embodiment is enumerated as follows:
A, strain and plasmid are that laboratory building saves.
(1) strain: E. coli BL21 (DE3) is the host strain of gene expression.
(2) plasmid: pET-22b (+)-ELP40.
B, major experimental reagent is the highest purity analyzing pure or market and capable of buying.
C, main medium formula
C.1 LB seed culture medium
LB culture medium: Tryptone 1.0g, Yeast Extract 0.5g, NaCl 1.0g, distilled water 100mL.
C.2 TB expands culture medium
Culture medium: Tryptone 12.0g, Yeast Extract 24.0g, Glycerol 4.5mL, NH4Cl 3.0g,
Distilled water 900mL is added to dissolve, 121 DEG C of high pressure sterilization 20min；
Phosphate solution: K2HPO4·12H2O 16.43g, KH2PO4 2.31g, distilled water 100mL；121 DEG C of high pressure sterilizations
20min；
The solution of above two sterilizing is cooled to room temperature, mixing is TB culture medium.
D, experimental solutions are prepared
(1) 100mg/mL ampicillin sodium: ampicillin sodium 1.00g is weighed, ddH is dissolved in2O is settled to 10mL, uses
0.22 μm of membrane filtration degerming after packing, is saved in -20 DEG C.
(2) IPTG of 100mmol/L: IPTG 2.38g is weighed, ddH is dissolved in2O is settled to 100mL, with 0.22 μm of filter
Film filtration sterilization after packing, is kept in dark place in -20 DEG C.
(3) buffer:
I PBS buffer solution:
A liquid (0.2mol/L disodium phosphate soln): Na is weighed2HPO4·12H2O 71.64g, is dissolved in distilled water, fixed
Hold to 1000mL.
B liquid (0.2mol/L sodium dihydrogen phosphate): Na is weighed2HPO424.0g is dissolved in distilled water, is settled to
1000mL。
1 0.2mol/L PBS buffer solution of table
A, B liquid are mixed according to the ratio in Table 1, and adding isometric distilled water is the PBS buffer solution of 0.1mol/L difference pH value.
II disodium hydrogen phosphate-citrate buffer solution:
A liquid (0.2mol/L Na2HPO4): weigh Na2HPO4·12H2O 71.64g, is dissolved in deionized water, is settled to
1000mL。
B liquid (0.1mol/L citric acid): C is weighed6H8O7·H2O 21.01g, is dissolved in deionized water, is settled to
1000mL。
2 0.1mol/L citric acid of table and 0.2mol/L disodium hydrogen phosphate buffer
A, B liquid, disodium hydrogen phosphate-citrate buffer solution of as different pH are mixed in 2 ratio of table.
The standard solution of the silica of the 100 μ g/mL with 100mL: take the silica of 1000 μ g/mL molten with liquid-transfering gun
Liquid 10mL, with 0.05M sodium hydroxide solution constant volume in 100mL volumetric flask.
By table 3, with liquid-transfering gun by 100 μ g/mL, then 0.05M sodium hydroxide solution constant volume 10mL, is made into various concentration
Silicon dioxde solution.
The silicon dioxde solution of 3 various concentration of table
(6) preparation of 1mmol/L hydrochloric acid: first compound concentration 1mol/L takes the hydrochloric acid of 8.6mL 36% to add water, stirring, determine
Hold to 100mL.The 1mmol/L hydrochloric acid of 1mL is taken, dilutes, is settled to 100mL.
(7) preparation of the silicate solution of 1mol/L: the tetramethylsilane (TMOS) of 1.522g is weighed, with the salt of 1mmol/L
Acid dissolution is settled to 10mL, obtains the silicate solution of Fresh, is stored at room temperature 15min, can be directly as silicon source.Silicic acid
It is preferably ready-to-use.
In addition, the application about the unaccomplished matter of experimental material see inventor deliver the same period (including instruction of papil hair
Table) other articles or paper obtain.
The application also provides a kind of application of Nano particles of silicon dioxide prepared using above-mentioned preparation method.The application system
Standby Nano particles of silicon dioxide is concentrated because of its uniform particle sizes, is had extraordinary characteristic, be can be widely applied to immobilised enzymes
Carrier, enzyme self-retaining is the process that enzyme spontaneously forms insoluble state enzyme under certain condition or in medium, such as
By ELP40 and zytase amalgamation and expression, the self-retaining of the zytase of silicon substrate is realized.For another example bionical silica is received
The carrier of slow releasing pharmaceutical etc. that rice corpuscles is used as.
It should be emphasized that term "comprises/comprising" refers to the presence of feature, element, step or component when using herein, but simultaneously
It is not excluded for the presence or additional of one or more other features, element, step or component.
In addition, method of the invention be not limited to specifications described in time sequencing execute, can also according to it
His time sequencing, concurrently or independently execute.Therefore, the execution sequence of method described in this specification is not to this hair
Bright technical scope is construed as limiting.
Although being had been disclosed above by the description to specific embodiments of the present invention to the present invention, it answers
The understanding, above-mentioned all embodiments and example are exemplary, and not restrictive.Those skilled in the art can be in institute
Design is to various modifications of the invention, improvement or equivalent in attached spirit and scope of the claims.These modification, improve or
Person's equivalent should also be as being to be considered as included in protection scope of the present invention.
Claims (10)
1. a kind of preparation method for the bionic Nano particles of silicon dioxide that polypeptide mediates, characterized by the following steps:
Step 1: using the class elastin polypeptide ELPs of ITC method of purification preparation high-purity；ITC method of purification specifically includes: first
The heating of ELPs solution is allowed to undergo phase transition, centrifugation is gone to collect and be precipitated under the conditions of being higher than Tt；Then, molten with the buffer of pre-cooling
Solution precipitating, is centrifuged under cryogenic, carries out renaturation process to the precipitating after centrifugation, collects supernatant, this is the class bullet purified
Property polypeptide ELPs；
Step 2: class elastin polypeptide ELPs mediates silicon dioxide granule precipitating, obtains bionic Nano particles of silicon dioxide；Tool
Body comprises the following processes:
The class elastin polypeptide ELPs that step 1 obtains is dissolved in PBS buffer solution and obtains ELPs solution, class elastin polypeptide
The concentration range of ELPs is 10 μm of ol/L-100 μm of ol/L, pH=6.9-7.1；
By SiO2Precursor is dissolved in preparation silicon source solution, SiO in solution2Precursor is tetramethylsilane or tetraethyl silane；
The ELPs solution that volume is a is mixed with the silicon source solution that volume is b, a: b=(9-11): 1；
Stand preset time at room temperature after mixing, high speed centrifugation collects precipitation of silica to get bionical titanium dioxide is arrived
Silicon nano.
2. the preparation method of bionic Nano particles of silicon dioxide according to claim 1, it is characterised in that: silicon source solution is matched
Process processed is as follows:
Prepare initial silicon source solution: the SiO for being c by quality2Precursor is dissolved in the hydrochloric acid that concentration is d, and constant volume v can be obtained initially
Silicon source solution；C unit be gram, d unit be mmol/L, v unit be mL；
Prepare discrete graphite solution: it is in the hydrochloric acid of d that the graphite that quality is e, which is dissolved in concentration, wherein c > 5e, constant volume v, then
It carries out ultrasound and the discrete graphite solution of acquisition is blended；E unit is gram；
Then initial silicon source solution and discrete graphite solution are mixed into simultaneously high-speed stirred preset time, mixed silicon can be obtained
Source solution.
3. the preparation method of bionic Nano particles of silicon dioxide according to claim 2, it is characterised in that: after wherein mixing
Silicon source solution obtained by following process: hydrochloric acid is added in graphite, carries out ultrasonic agitation preset time, obtains suspension, so
Suspension and initial silicon source solution are quickly mixed afterwards, are stirred preset time at 35-60 DEG C, mixed silicon can be obtained
Source solution.
4. the preparation method of bionic Nano particles of silicon dioxide according to claim 2, it is characterised in that: what is obtained is bionical
The size of Nano particles of silicon dioxide is 70nm to 400nm, the quality percentage of silica in bionic Nano particles of silicon dioxide
Than for 10%-99%.
5. the preparation method of bionic Nano particles of silicon dioxide according to claim 2, it is characterised in that: the quality of graphite
Percent concentration ω range is 3wt%-3.7wt%, ω=quality e*10-3/12*v。
6. the preparation method of bionic Nano particles of silicon dioxide according to claim 1, it is characterised in that: step 1 obtains
Class elastin polypeptide ELPs gene order be ELP [KV8F-s], ELP [KV8F-s] refer to continuous 10 VPGXG pentapeptides
In unit, the 4th Xaa of each pentapeptide has 1 lysine (K), 8 valines (V), 1 phenylalanine (F), and s refers to there is s
It is a using VPGXG as the repetitive sequence of unit, the value of s is 10-300.
7. the preparation method of bionic Nano particles of silicon dioxide according to claim 1, it is characterised in that: right in step 1
Precipitating after centrifugation carries out renaturation process and specifically includes: carrying out fast dewatering to being deposited within the scope of -18 DEG C to 0 DEG C of temperature first
Processing, and it is shatter to the precipitating progress appropriateness after dehydration, particulate solid is obtained, then the particulate solid was carried out
Sieve, selection by winnowing or high-pressure electrostatic absorption, most of inclusion body are removed, the precipitating after obtaining renaturation.
8. the preparation method of bionic Nano particles of silicon dioxide according to claim 7, it is characterised in that: at the renaturation
Reason further include: refolding is at low temperature to reduce the formation of protein aggregation by the precipitating after renaturation, when forming aggregation
When intermediate is reduced, temperature is improved 10 degree rapidly, to promote protein folding renaturation, to can get high annealing efficiency
Solid granulates；Low temperature refers to -18 DEG C to -10 DEG C.
9. a kind of application of the bionic Nano particles of silicon dioxide obtained by any preparation method of claim 1-8, described bionical
Nano particles of silicon dioxide is used for the self-retaining of enzyme.
10. a kind of application of the bionic Nano particles of silicon dioxide obtained by any preparation method of claim 1-8, described bionical
The carrier for the slow releasing pharmaceutical that Nano particles of silicon dioxide is used as.
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