CN103451165B - 3’,5’-二磷酸腺苷酸特异性3’-核苷酸酶及其构建方法和应用 - Google Patents
3’,5’-二磷酸腺苷酸特异性3’-核苷酸酶及其构建方法和应用 Download PDFInfo
- Publication number
- CN103451165B CN103451165B CN201310339369.0A CN201310339369A CN103451165B CN 103451165 B CN103451165 B CN 103451165B CN 201310339369 A CN201310339369 A CN 201310339369A CN 103451165 B CN103451165 B CN 103451165B
- Authority
- CN
- China
- Prior art keywords
- phosphonuclease
- seq
- specificity
- sequence
- protein
- 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.)
- Active
Links
- 239000002126 C01EB10 - Adenosine Substances 0.000 title claims abstract description 11
- 229960005305 adenosine Drugs 0.000 title claims abstract description 11
- 238000000034 method Methods 0.000 title claims description 36
- 238000010276 construction Methods 0.000 title claims description 6
- 230000008569 process Effects 0.000 title description 5
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 27
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 14
- 235000018102 proteins Nutrition 0.000 claims abstract description 12
- 241000894006 Bacteria Species 0.000 claims abstract description 10
- 238000001514 detection method Methods 0.000 claims abstract description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 4
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 claims description 16
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 claims description 15
- 230000008859 change Effects 0.000 claims description 10
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 7
- 230000002018 overexpression Effects 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000013461 design Methods 0.000 claims description 4
- 238000002703 mutagenesis Methods 0.000 claims description 4
- 231100000350 mutagenesis Toxicity 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 108091008146 restriction endonucleases Proteins 0.000 claims description 4
- 239000013598 vector Substances 0.000 claims description 4
- 102000003960 Ligases Human genes 0.000 claims description 3
- 108090000364 Ligases Proteins 0.000 claims description 3
- 239000011543 agarose gel Substances 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 239000006166 lysate Substances 0.000 claims description 3
- 230000000869 mutational effect Effects 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 235000015097 nutrients Nutrition 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 238000012795 verification Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 102100035703 Prostatic acid phosphatase Human genes 0.000 abstract description 29
- 230000000694 effects Effects 0.000 abstract description 14
- 150000001413 amino acids Chemical class 0.000 abstract description 12
- 235000001014 amino acid Nutrition 0.000 abstract description 9
- 102000004357 Transferases Human genes 0.000 abstract description 8
- 108090000992 Transferases Proteins 0.000 abstract description 8
- 238000004458 analytical method Methods 0.000 abstract description 6
- 230000002255 enzymatic effect Effects 0.000 abstract description 6
- 241000193830 Bacillus <bacterium> Species 0.000 abstract description 4
- 108010032655 Adenylyl-sulfate reductase Proteins 0.000 abstract description 3
- 238000001228 spectrum Methods 0.000 abstract description 3
- 240000007594 Oryza sativa Species 0.000 abstract description 2
- 235000007164 Oryza sativa Nutrition 0.000 abstract description 2
- 108010043671 prostatic acid phosphatase Proteins 0.000 abstract description 2
- 235000009566 rice Nutrition 0.000 abstract description 2
- 241000894007 species Species 0.000 abstract description 2
- 102000004008 5'-Nucleotidase Human genes 0.000 abstract 1
- GACDQMDRPRGCTN-KQYNXXCUSA-N 3'-phospho-5'-adenylyl sulfate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OS(O)(=O)=O)[C@@H](OP(O)(O)=O)[C@H]1O GACDQMDRPRGCTN-KQYNXXCUSA-N 0.000 description 30
- WHTCPDAXWFLDIH-UHFFFAOYSA-N PAP Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(OP(O)(O)=O)C1O WHTCPDAXWFLDIH-UHFFFAOYSA-N 0.000 description 29
- 101150105093 PaPS gene Proteins 0.000 description 27
- 102000004190 Enzymes Human genes 0.000 description 21
- 108090000790 Enzymes Proteins 0.000 description 21
- 239000002253 acid Substances 0.000 description 13
- -1 nucleoside diphosphate Chemical class 0.000 description 13
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 12
- 239000001177 diphosphate Substances 0.000 description 12
- 235000011180 diphosphates Nutrition 0.000 description 12
- 239000002777 nucleoside Substances 0.000 description 12
- IRLPACMLTUPBCL-KQYNXXCUSA-N 5'-adenylyl sulfate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OS(O)(=O)=O)[C@@H](O)[C@H]1O IRLPACMLTUPBCL-KQYNXXCUSA-N 0.000 description 11
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 10
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical group C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000013016 damping Methods 0.000 description 9
- 239000012530 fluid Substances 0.000 description 9
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 9
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 9
- 229950006790 adenosine phosphate Drugs 0.000 description 8
- 239000000758 substrate Substances 0.000 description 8
- DTBNBXWJWCWCIK-UHFFFAOYSA-N phosphoenolpyruvic acid Chemical compound OC(=O)C(=C)OP(O)(O)=O DTBNBXWJWCWCIK-UHFFFAOYSA-N 0.000 description 6
- 108090000765 processed proteins & peptides Proteins 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 102000002281 Adenylate kinase Human genes 0.000 description 5
- 108020000543 Adenylate kinase Proteins 0.000 description 5
- 241000282326 Felis catus Species 0.000 description 5
- 230000003197 catalytic effect Effects 0.000 description 5
- 238000006555 catalytic reaction Methods 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 108010022348 Sulfate adenylyltransferase Proteins 0.000 description 4
- 230000026731 phosphorylation Effects 0.000 description 4
- 238000006366 phosphorylation reaction Methods 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 238000002798 spectrophotometry method Methods 0.000 description 4
- 108010071218 3'-phosphoadenylyl-5'-phosphosulfate reductase Proteins 0.000 description 3
- 101100270014 Arabidopsis thaliana APR2 gene Proteins 0.000 description 3
- 108020005115 Pyruvate Kinase Proteins 0.000 description 3
- 102000013009 Pyruvate Kinase Human genes 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 108010028584 nucleotidase Proteins 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000001180 sulfating effect Effects 0.000 description 3
- LSNNMFCWUKXFEE-UHFFFAOYSA-L sulfite Chemical compound [O-]S([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-L 0.000 description 3
- 102100040149 Adenylyl-sulfate kinase Human genes 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101100202428 Neopyropia yezoensis atps gene Proteins 0.000 description 2
- 102000004523 Sulfate Adenylyltransferase Human genes 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 239000005864 Sulphur Substances 0.000 description 2
- 102100036407 Thioredoxin Human genes 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 229940011871 estrogen Drugs 0.000 description 2
- 239000000262 estrogen Substances 0.000 description 2
- 238000005558 fluorometry Methods 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 238000009413 insulation Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 230000033001 locomotion Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 229940107700 pyruvic acid Drugs 0.000 description 2
- 230000018612 quorum sensing Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000019635 sulfation Effects 0.000 description 2
- 238000005670 sulfation reaction Methods 0.000 description 2
- 108060008226 thioredoxin Proteins 0.000 description 2
- 229940094937 thioredoxin Drugs 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- HWKRAUXFMLQKLS-UHFFFAOYSA-N 2-oxidanylidenepropanoic acid Chemical compound CC(=O)C(O)=O.CC(=O)C(O)=O HWKRAUXFMLQKLS-UHFFFAOYSA-N 0.000 description 1
- 102100022464 5'-nucleotidase Human genes 0.000 description 1
- AEOBEOJCBAYXBA-UHFFFAOYSA-N A2P5P Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1OP(O)(O)=O AEOBEOJCBAYXBA-UHFFFAOYSA-N 0.000 description 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 description 1
- 102100032145 Carbohydrate sulfotransferase 10 Human genes 0.000 description 1
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101000775595 Homo sapiens Carbohydrate sulfotransferase 10 Proteins 0.000 description 1
- 101000585332 Homo sapiens Sulfotransferase 1C4 Proteins 0.000 description 1
- 108010025815 Kanamycin Kinase Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 102000003939 Membrane transport proteins Human genes 0.000 description 1
- 108090000301 Membrane transport proteins Proteins 0.000 description 1
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 230000010748 Photoabsorption Effects 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 101710188306 Protein Y Proteins 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 102100029863 Sulfotransferase 1C4 Human genes 0.000 description 1
- 102000004896 Sulfotransferases Human genes 0.000 description 1
- 108090001033 Sulfotransferases Proteins 0.000 description 1
- 108010064721 Type I Secretion Systems Proteins 0.000 description 1
- 101150080168 XA21 gene Proteins 0.000 description 1
- 241000589634 Xanthomonas Species 0.000 description 1
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000000516 activation analysis Methods 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- WHTCPDAXWFLDIH-KQYNXXCUSA-N adenosine 3',5'-bismonophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H]1O WHTCPDAXWFLDIH-KQYNXXCUSA-N 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000032770 biofilm formation Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 239000012145 high-salt buffer Substances 0.000 description 1
- 235000014304 histidine Nutrition 0.000 description 1
- 150000002411 histidines Chemical class 0.000 description 1
- 108010087116 holo-(acyl-carrier-protein) synthase Proteins 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 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
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 108010091212 pepstatin Proteins 0.000 description 1
- FAXGPCHRFPCXOO-LXTPJMTPSA-N pepstatin A Chemical compound OC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)CC(C)C FAXGPCHRFPCXOO-LXTPJMTPSA-N 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 229930029653 phosphoenolpyruvate Natural products 0.000 description 1
- UEZVMMHDMIWARA-UHFFFAOYSA-M phosphonate Chemical group [O-]P(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-M 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229940076788 pyruvate Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- QYHFIVBSNOWOCQ-UHFFFAOYSA-N selenic acid Chemical compound O[Se](O)(=O)=O QYHFIVBSNOWOCQ-UHFFFAOYSA-N 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 229960004249 sodium acetate Drugs 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 150000003464 sulfur compounds Chemical class 0.000 description 1
- 238000004481 total suppression of sideband Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
本发明涉及一种特异性3’-核苷酸酶。3’,5’-二磷酸腺苷酸特异性3’-核苷酸酶,其为以下(a)或(b)的蛋白质:(a)由SEQ ID:NO.5所示的氨基酸序列组成的蛋白质;(b)在(a)的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有3’,5’-二磷酸腺苷酸特异性3’-核苷酸酶活性的由(a)衍生的蛋白质。本发明构建了一系列的G236突变体,通过酶学分析发现本发明获得了PAP专一性的3’核苷酸酶。本发明进一步利用此突变体,对来自水稻白叶枯病菌的硫酸转移酶RaxST以及结核杆菌APS还原酶(tbAPSR)进行了测定、分析,提高了检测的效率。
Description
技术领域
本发明涉及一种特异性3’-核苷酸酶。
背景技术
硫作为构成生命体的大量元素之一,是生命活动的必需元素。硫酸根离子通过膜转运蛋白进入细胞后,在ATP硫酸化酶(ATP sulfurylase,ATPS)的催化下接受ATP的腺苷一磷酸基团形成腺苷-5’-磷酰硫酸(adenosine 5’-phosphosulfate,APS)。APS的3’羟基可被APS激酶(APS kinase,APSK)磷酸化形成3’-磷酸-腺苷-5’-磷酰硫酸(3’-phosphoadenosine 5’-phosphosulfate, PAPS),PAPS可被PAPS还原酶等还原,最后整合到半胱氨酸及其它还原性硫化合物中(Leyh, TS,Crit Rev Biochem Mol Biol,1993,28, 515-42;王艳兴等,生命的化学,2011,31(2): 252-257),也可作为硫酸转移酶(sulfotranferase,ST)的底物进行硫酸化反应,参与化合物的生理功能调节及蛋白之间的互作(Leyh, TS,Crit Rev Biochem Mol Biol,1993,28, 515-42;Liu等,IUBMB Life,2007,59(10): 622-627)(表1)。
3’,5’二磷酸腺苷(3’-phosphoadenosine 5’-phosphate,PAP)在细胞内是硫代谢的中间代谢物之一(Leyh, TS,Crit Rev Biochem Mol Biol,1993,28, 515-42),如PAPS还原酶催化PAPS的还原生成PAP和亚硫酸盐;硫酸转移酶催化来自3’-磷酸-腺苷-5’-磷酰硫酸(3’-phosphoadenosine 5’-phosphosulfate,PAPS)的活化硫酸根转移至其他化合物生成PAP;此外,非酶催化的3’-磷酸-腺苷-5’-磷酰硒酸(3’-phosphoadenosine 5’-phosphosulfate,PAPSe)的水解也产生PAP(Yu 等, Arch Biochem Biophys 1989,269: 156-174; Hanna 等,2004,J Biol Chem 279, 4415-4424)。PAP在细胞内,可受到3’,5’二磷酸核苷酸酶(3’,5’-bisphosphate nucleotidase,ND)(Murguia等,J Biol Chem 1996,271(46): 29029-29033;Murguia等,Science 1995,267(5195): 232-234;Albert等,J Mol Biol 2000,295(4): 927-938.)催化3’-磷酸基团的水解产生5’单磷酸腺苷(adenosine 5’-monophosphate,AMP),其Km为0.7~17 μM (Albert 等,J Mol Biol 2000,295(4): 927-938;Spiegelberg等,J Biol Chem 1999,274(19): 13619-13628;Aggarwal 等,Arch Biochem Biophys 2008,469(2): 174-183)。AMP可经肌激酶(myokinase,MK)磷酸化生成5’二磷酸腺苷(adenosine 5’-diphosphate,ADP)以及丙酮酸激酶(pyruvate kinase,PK)的进一步磷酸化生成ATP(表1)。研究表明酵母ND(yeast 3’,5’-bisphosphate nucleotidase,YND)亦可水解PAPS生成腺苷-5’-磷酰硫酸(adenosine 5’-phosphosulfate,APS)和磷酸(Spiegelberg等,J Biol Chem 1999,274(19): 13619-13628)。ND活性降低引起胞内PAP累积(Murguia等,J Biol Chem 1996,271(46): 29029-29033;Murguia 等,Science 1995,267(5195): 232-234;Albert 等,J Mol Biol 2000,295(4): 927-938;Spiegelberg 等,J Biol Chem 1999,274(19): 13619-13628;Mechold 等,Nucleic Acids Res 2006,34(8): 2364-2373),可影响硫酸转移酶(sulfotransferase,ST)(Whittemore等,Biochemistry 1985,24(10): 2477-2482;Ramaswamy等,J Biol Chem 1987,262(21): 10044-10047), 酰基载体蛋白合酶(Yang等,Eur J Biochem 1994,224(2): 743-750)以及RNA加工酶(Dichtl等,EMBO J 1997,16(23): 7184-7195;Mechold等,Nucleic Acids Res 2006,34(8): 2364-2373)的活性,影响细胞的生长发育。
表1,本发明中提及的主要反应式
反应 | 催化剂 |
PAPS + thioredoxin = PAP + SO3 2- + thioredoxin disulfide | PAPS还原酶 |
PAPS + X-OH = PAP + X-SO4 2- | 硫酸转移酶 |
PAP = AMP + Pi | 3’,5’二磷酸核苷酸酶 |
PAPS = APS + Pi | 3’,5’二磷酸核苷酸酶 |
AMP + ATP = 2 ADP | 肌激酶 |
ADP + phosphoenolpyruvate = ATP + pyruvate | 丙酮酸激酶 |
Pyruvate + NADH = NAD+ + lactate | 乳酸脱氢酶 |
PAPS + axY22 = PAP + axYS22 | RaxST |
ATP + SeO4 2- = APSe + PPi ATP + SO4 2- = APS + PPi | ATPS |
ATP + APSe = PAPSe + ADP ATP + APS = PAPS + ADP | APSK |
目前,ST的活性测定方法包括:同位素标记法(PAP35S或者H3底物等)、分光光度法、荧光分析法以及质谱分子法;这些方法可分为连续测定和非连续测定,如同位素标记法和质谱分子法主要是非连续测定,而分光光度法、荧光分析法主要是连续测定(Paul P, Anal Bioanal Chem, 2012,403: 1491)。这些方法各有其优缺点,如同位素标记法虽然很灵敏,但其准确性、非连续性及对操作人员的毒害作用等,使得其不是最理想的方法;质谱分析法的主要优点是准确、低毒、可分析碳水化合物等具有多个硫酸化位点的反应,缺点主要是仪器昂贵和测定的非连续性;分光光度法和荧光光度法相对经济、高通量、准确和操作方便,缺点为并不是所有的底物或者产物有光吸收或者荧光特性,所以可通过产物与具有相关特性的反应偶联,完成此种测定。Prather等(Anal Biochem,2012,423: 86)利用高尔基体PAP特异的3’核苷酸酶(Km,kcat分别为98.2mM和11.3s-1)和分光光度磷酸盐检测方法,连续测定了CHST10和SULT1C4两种ST的活性;Han等(Nat Commun. 2012,3:1153)利用非连续分步分光光度法测定RaxST反应底物PAPS的减少,进而分析ST的活性。
发明内容
为了解决上述的技术问题,本发明的第一个目的是提供一种3’,5’-二磷酸腺苷酸特异性3’-核苷酸酶,该特异性3’-核苷酸酶可作为PAP检测用高效偶联酶。本发明的第二个目的是提供编码上述的特异性3’-核苷酸酶的基因。本发明的第三个目的是提供一种构建上述的特异性3’-核苷酸酶的方法。本发明的第四个目的是上述的特异性3’-核苷酸酶的应用。
为了实现上述的第一个目的,本发明采用了以下的技术方案:
3’,5’-二磷酸腺苷酸特异性3’-核苷酸酶,其为以下(a)或(b)的蛋白质:
(a)由SEQ ID:NO.5所示的氨基酸序列组成的蛋白质;
(b)在(a)的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有3’,5’-二磷酸腺苷酸特异性3’-核苷酸酶活性的由(a)衍生的蛋白质。
作为优选,所述的(b)的氨基酸序列为SEQ ID:NO.6 、SEQ ID:NO.7或SEQ ID:NO.8所示。
为了实现上述的第二个目的,本发明采用了以下的技术方案:
编码上述的蛋白质的基因。
作为优选,该基因的核苷酸序列为SEQ ID:NO.1、SEQ ID:NO.2 、SEQ ID:NO.3或SEQ ID:NO.4所示的DNA分子。
为了实现上述的第三个目的,本发明采用了以下的技术方案:
一种构建权利要求1所述的3’,5’-二磷酸腺苷酸特异性3’-核苷酸酶的方法,该方法包括以下的步骤:
1)PCR法克隆的YND核苷酸序列和pET24a原核蛋白超表达载体,分别经限制性内切酶Nde I/Hind III双酶切后,利用T4连接酶连接构建表达载体pYND,用于表达YND;
2)利用PCR突变的方法获得DNA:根据YND基因的序列与所要突变的位点,设计定点突变PCR引物,以突变位点为中心,左右各取10-15个碱基,引物长度为25-45个碱基;
3)所获DNA转化E. coli DH5α 感受态细胞扩增质粒并测序验证;
4)将突变质粒转化E. coli BL21,接种过夜培养的菌液于液体培养基中培养,收集菌体;
5)取裂解液,悬浮菌体,混合均匀后,冰浴超声波破碎菌体,离心收集上清;
6)利用PAP琼脂糖凝胶纯化YND及其突变蛋白。
作为优选,所述的突变PCR引物的序列如下:
引物序列(5’-3’) | |
G236A1 | CATGATTACTTTAGAGGCAGTTGAAAAGGGACACTC |
G236A2 | GAGTGTCCCTTTTCAACTGCCTCTAAAGTAATCATG |
G236C1 | CATGATTACTTTAGAGTGCGTTGAAAAGGGACACTC |
G236C2 | GAGTGTCCCTTTTCAACGCACTCTAAAGTAATCATG |
G236V1 | CATGATTACTTTAGAGGTCGTTGAAAAGGGACACTC |
G236V2 | GAGTGTCCCTTTTCAACGACCTCTAAAGTAATCATG |
G236S1 | CATGATTACTTTAGAGAGCGTTGAAAAGGGACACTC |
G236S2 | GAGTGTCCCTTTTCAACGCTCTCTAAAGTAATcatCATG |
为了实现上述的第四个目的,本发明采用了以下的技术方案:
上述的特异性3’-核苷酸酶用于3’,5’二磷酸腺苷的检测。
本发明由于采用了上述的技术方案,通过分析YND的晶体结构(www.pdb.org;PDB:1KA1),发现氨基酸的236位(甘氨酸)小R基团是H,G236的小R基团,可能有利于PAPS的结合。将其变为其他较大的氨基酸,有可能影响对底物PAP及PAPS的结合。本发明构建了一系列的G236突变体,通过酶学分析发现本发明获得了PAP专一性的3’核苷酸酶。本发明进一步利用此突变体,对来自水稻白叶枯病菌的硫酸转移酶RaxST以及结核杆菌APS还原酶(tbAPSR)进行了测定、分析,提高了检测的效率。
附图说明
图1为tbAPSR还原PAPS的活性分析图。
图2为RaxSTKm PAPS的测定图。
图3为RaxSTKm axY22的测定图。
具体实施方式
实施例1 特异性3’-核苷酸酶的构建
PCR法克隆的酵母菌3’,5’二磷酸核苷酸酶(yeast 3’,5’-bisphosphate nucleotidase,YND)(正向引物:GGAATTCCATATGCACCACCACCACCACCACGCATTGGAAAGAGAATTATTGG, 反向引物:AAGCTTGGCGTTTCTTGACTGAATGAC)核苷酸序列和pET24a原核蛋白超表达载体,分别经限制性内切酶Nde I/Hind III双酶切后,利用T4连接酶连接构建表达载体pYND,用于表达YND。
根据YND基因的序列(www.pdb.org;PDB:1KA1)与所要突变的位点,按照Stratagene 的Site-Directed Mutagenesis Kit 引物设计方法,设计定点突变PCR引物,以突变位点为中心,左右各取10-15 个碱基,引物长度一般为25-45 个碱基(表2)。突变利用PCR的方法进行,依次向无菌PCR 管中加入以下试剂:ddH2O 32.5 μL,5×PrimeSTARTM Buffer 10 μL,dNTPs Mix(各2.5 mM) 4 μL,正向引物(100 ng/μL) 1.25 μL,反向引物(100 ng/μL) 1.25μL,pYND质粒载体(100 ng/μL) 0.5 μL,PrimeSTAR DNA Polymerase(2.5 U/μL) 0.5 μL;混匀后短暂离心,按PCR程序(98℃ 变性5 min,98℃变性15 sec,60℃退火15 sec,72℃延伸8 min,进行20个循环后72℃保温10 min)进行扩增(Bio-Rad MyCycler)。
表2 定点突变引物
引物名称 | 引物序列(5’-3’) |
G236A1 | Catgattactttagaggcagttgaaaagggacactc |
G236A2 | GAGTGTCCCTTTTCAACTGCCTCTAAAGTAATCATG |
G236C1 | Catgattactttagagtgcgttgaaaagggacactc |
G236C2 | Gagtgtcccttttcaacgcactctaaagtaatcatg |
G236V1 | Catgattactttagaggtcgttgaaaagggacactc |
G236V2 | GAGTGTCCCTTTTCAACGACCTCTAAAGTAATCATG |
G236S1 | CatgattactttagagAGCgttgaaaagggacactc |
向PCR扩增产物中加入1 μL Dpn I 限制性酶,37℃温浴1 h,以消化模板DNA;将Dpn I 处理后的PCR 产物加灭菌的ddH2O 稀释到200 μL,加入等体积的氯仿:异戊醇(24:1)上下颠倒混匀,12000 rpm 离心10 min。取上部水相,移入1.5 mL 离心管中,加1/10 体积3 M 醋酸钠(pH 5.2),混匀后加入2.5 倍体积预冷的无水乙醇,混匀后-20℃保温10-20 min后12000 rpm,离心10 min;弃上清,用75%乙醇洗涤两次,晾干后加20 μL ddH2O溶解DNA。所获DNA转化E. coli DH5α 感受态细胞扩增质粒并测序验证。
将突变质粒转化E. coli BL21(DE3),接种过夜培养的菌液于500 mL 含Amp 的LB 液体培养基中,37℃振荡培养至 OD600 为0.6~0.8;将菌液冷却至16℃,加入0.5 mM IPTG,继续培养16 h。5000 rpm 离心5 min,收集菌体。
取20 mL 裂解液(50 mM KCl、10 mM CaCl2、5 mM EDTA,1 mM DTT、290 μM PMSF、1.5 μM Pepstatin A、0.1 mg/mL 溶菌酶、50 mM Hepes 缓冲液,pH 8.0),悬浮菌体,混合均匀后,于4℃放置1 h,其间颠倒混匀数次,以免菌体沉淀。冰浴超声波破碎菌体,超声5 sec,间隔10 sec,重复超声约200 次。20000×g 于4℃离心30 min,收集上清。
利用PAP琼脂糖凝胶纯化YND及其突变蛋白。含目的蛋白上清液经1 mL/min的流速上样至PAP琼脂糖凝胶柱,分别经5倍柱体积高盐缓冲液(4 M NaCl、10 mM CaCl2、50 mM Hepes 缓冲液,pH 8.0)、5倍柱体积低盐缓冲液(50 mM NaCl、10 mM CaCl2、50 mM Hepes 缓冲液,pH 8.0)洗涤后,利用洗脱液(50 mM KCl、5 mM EGTA、50 mM Hepes 缓冲液,pH 8.0)将目的蛋白洗脱下来,并经透析(50 mM Hepes 缓冲液,pH 8.0),获得蛋白YND,G236S,G236V,G236A和 G236C,-80℃冰箱保存待用。
实施例2 特异性3’-核苷酸酶的活性测定
利用MK和PK/LDH偶联酶系统(Rhoads等,J. Biol. Chem. 1968, 243:3963-3972)测定YND及其突变蛋白对PAP的水解活性。生成的ADP可通过PK/LDH体系(Malcovati等,Methods Enzymol,1982,90:170-179)中的PK磷酸化生成ATP和丙酮酸,丙酮酸可进一步被LDH通过氧化NADH(e339nm = 0.622 mM-1×cm-1)而还原为乳酸。测定条件为:50 mM Hepes缓冲液(pH 8.0);50 mM KCl;0.25mM NADH;10 U/mL PK;25 mM PEP;20 U/mL LDH;2 mM MgCl2;50 nM YND或者突变蛋白;2 U/mL MK;1.0 mM ATP;利用3mM PAP起始反应。利用荧光分光光度计连续测定NADH的氧化速率,利用连续曲线法(Tang等,J Phys Chem. 2010,114:16131-16136)计算酶活。
利用APSK和PK/LDH偶联酶系统(Sun等,J. Biol. Chem. 2005, 280:7861–7866)测定YND及其突变蛋白对PAPS的水解活性。生成的ADP进一步利用PK/LDH体系进行测定。测定条件为: 50 mM Hepes缓冲液(pH 8.0);50 mM KCl;0.25mM NADH;10 U/mL PK;25 mM PEP;20 U/mL LDH;2 mM MgCl2;50 nM YND或者突变蛋白;2 U/mL APSK;1.0 mM ATP;不同浓度的PAPS起始反应。利用紫外可见分光光度计连续测定NADH的氧化速率,计算酶活。 经考马斯亮蓝法定量YND及突变蛋白浓度,通过酶偶联系统,分别测定突变蛋白的PAP/PAPS的底物的米氏常数,获得相应的kcat,Km(表3)。
表3.不同突变体的底物特异性分析
Units for kcat,Km and kcat/Km are s-1,μM and ′106 M-1s-1,respectively.
如表3所示,不同突变体中,G236V对PAP和PAPS的催化效率差异最大,对PAP的催化效率为1.8′107 M-1s-1,是对PAPS催化效率的6700多倍。高尔基体来源的PAP特异性的3’核苷酸酶kcat,Km分别为11.3 s-1,98.2 μM(Prather等,Anal Biochem, 2012, 423:86-92),催化效率为1.1′105 M-1s-1。G236V的催化效率是高尔基体来源的PAP特异性的3’核苷酸酶的160倍。G236V蛋白可作为PAP检测用高效偶联酶。
实施例3 利用G236V蛋白测定结核杆菌APS还原酶(tbAPSR)还原PAPS的活性
APSR还原硫酸根形成亚硫酸根离子,是细胞同化硫酸盐的重要步骤之一。结核杆菌的APSR可还原APS生成AMP和亚硫酸盐,也可还原PAPS生成PAP和亚硫酸盐(Sun等,Biochemistry 2006, 45, 11304-11311;Carroll 等, PLoS Biol, 2005,3(8): e250)。SUN等(Biochemistry 2006, 45, 11304-11311)纯化分析了tbAPSR,并利用雌激素硫酸转移酶作为偶联酶测定了tbAPSR还原PAPS的活性,结果表明其kcat和Km分别为:0.7(±0.3) min-1和31(±3) mM。
本发明利用纯化的tbAPSR,利用G236V为偶联酶将其产物PAP转化为AMP,经MK/PK/LDH的检测体系,可计算PAP的生成速度。利用下述条件:0.54mM APSR,50mM Hepes(pH 8.0),50mM KCl,2mM MgCl2,1mM DTT,5mM GSH,30mM E.coli thioredoxin,2mM PEP,2mM ATP,10U/mL PK,20U/mL LDH,20U/mL MK,0.2mM NADH,5U/mL G236V,分别加入底物PAPS 5,15,33,76,169,312和505μM起始反应。如图1所示,本发明可计算出其Vm和Km分别为:0.59(±0.02)mM/min (相应kcat =1.1min-1)和17.1mM。本发明的结果表明利用此方法和已发表的利用雌激素硫酸转移酶作为偶联酶测定结果是一致的。
上述的方法采用G236S, G236A和 G236C蛋白也具有相应的技术效果。
实施例4 G236V蛋白测定硫酸转移酶RaxST的活性
G236V蛋白测定ST的活性,可通过MK/PK/LDH偶联酶体系测定AMP的生成(Rhoads等,J Biol Chem, 1968, 243:3963-3972),也可通过磷酸检测体系(Nixon等,Anal Biochem. 1998,265(2):299-307;Okoh等,Biochemistry,2006,45(49):14764-71),测定PAP水解的产物之一磷酸基团。
Xa21是最早发现的白叶枯病抗性基因,具广谱抗性,其无毒基因ax21翻译产物Ax21(activator of XA21-mediated immunity)为194个氨基酸的蛋白质,包含外泌信号肽,硫酸化17肽axYS22,后续多肽链三个部分(Lee 等,Science,2009,326, 850-853)。Ax21是目前发现首个直接作用于宿主受体,引发抗性反应的群体感应因子,也是革兰氏阴性菌中首次发现的介导调控运动,生物膜形成,毒力相关基因表达的群体感应因子。研究发现, Ax21的成熟必须经N端第22位酪氨酸的硫酸化修饰(Lee 等, (2009) Science326, 850-853),I类分泌系统(type I secretion system,TOSS)分泌出胞外,蛋白酶切去后续多肽链等过程。Han等(Nat Commun. 2012,3:1153)克隆RaxST基因并利用大肠杆菌进行了超表达,利用不连续的测定系统结果表明原核超表达的RaxST其Km和Vm分别为1.7 uM,0.18 units/mg蛋白(kcat为5.6min-1)。
本发明利用新霉素磷酸转移酶(NPT II)基因启动子(Kp)5’端引物:GGATCC AGTTCTTGAAGTGGTGGCCTAACTAC (BamHⅠ)和Kp 3’端引物:GTCGAC AACACCCCTTGTATTACTGTTTATGT ( SalⅠ)及pET24a载体为模板克隆启动子Kp;利用RaxST的3’端引物:GTCGAC ATG CATCATCATCATCATCAT CCGCCGCCGACCAGCAT (SalⅠ)和5’端引物:AAGCTTTCAATGATGATGATGATGATGTACCAGCAGCGCTCCATG (HindⅢ)及细菌(Xanthomonas oryzae pv. Oryzae,PXO99)基因组DNA为模板克隆RaxST,并克隆入pET24a多克隆位点,构建同源重组载体pKp-RaxST(NPT II启动子-6xHistines-RaxST)。参照shen等(Mol. Microbiol. 2002,44:37-48)方法构建超表达RaxST的PXO99-RaxST菌株。通过培养此PXO99细菌,裂解细胞,利用Ni-NTA柱纯化含有6个组氨酸的RaxST蛋白。
利用RaxST(20.8 mM),17肽(axY22,0.6 mM),不同浓度的PAPS,50 mM Hepes缓冲液(pH 8.0);50 mM KCl;0.25mM NADH;10 U/mL PK;25 mM PEP;20 U/mL LDH;2 mM MgCl2;2 U/mL 肌激酶和1.0 mM ATP进行硫酸化反应(图2)。结果表明PAPS的Vm和Km分别为:12.4(±1.0)mM/min和0.65(±0.11) mM。
利用RaxST(20.8 mM),PAPS(0.8 mM),不同浓度的17肽(AENLSYNFVEGDYVRTP,axY22),50 mM Hepes缓冲液(pH 8.0);50 mM KCl;0.25mM NADH;10 U/mL PK;25 mM PEP;20 U/mL LDH;2 mM MgCl2;2 U/mL 肌激酶和1.0 mM ATP进行硫酸化反应(图3)。利用米氏方程拟合,结果发现17肽axY22的Vm和Km分别为:10.5(±0.5)mM/min和0.26(±0.04) mM。RaxST表达来源的不同,可能造成本测定结果与Han等(Nat Commun. 2012,3:1153)结果有一定区别。
上述的方法采用G236S, G236A和 G236C蛋白也具有相应的技术效果。
序列表
<110>浙江师范大学
<120>3’,5’-二磷酸腺苷酸特异性3’-核苷酸酶及其构建方法和应用
<160>8
<210> 1
<211> 1074
<212> DNA
<213> 酵母菌3’,5’二磷酸核苷酸酶
<400> 1
1 ATGGCATTGG AAAGAGAATT ATTGGTTGCA ACTCAAGCTG TACGAAAGGC GTCTTTATTG
61 ACTAAGAGAA TTCAATCTGA AGTGATTTCT CACAAGGACT CCACTACTAT TACCAAGAAT
121 GATAATTCTC CAGTAACCAC AGGTGATTAT GCTGCACAAA CGATCATCAT AAATGCTATC
181 AAGAGCAATT TTCCTGATGA TAAGGTAGTT GGTGAAGAAT CCTCATCAGG ATTGAGCGAC
241 GCATTCGTCT CAGGAATTTT AAACGAAATA AAAGCCAATG ACGAAGTTTA TAACAAGAAT
301 TATAAAAAGG ATGATTTTCT GTTTACAAAC GATCAGTTTC CGCTAAAATC TTTGGAGGAC
361 GTCAGGCAAA TCATCGATTT CGGCAATTAC GAAGGTGGTA GAAAAGGAAG ATTTTGGTGT
421 TTGGATCCTA TTGACGGAAC CAAGGGGTTT TTAAGAGGTG AACAGTTTGC AGTATGTCTG
481 GCCTTAATTG TGGACGGTGT TGTTCAGCTT GGTTGTATTG GATGCCCCAA CTTAGTTTTA
541 AGTTCTTATG GGGCCCAAGA TTTGAAAGGC CATGAGTCAT TTGGTTATAT CTTTCGTGCT
601 GTTAGAGGTT TAGGTGCCTT CTATTCTCCA TCTTCAGATG CAGAGTCATG GACCAAAATC
661 CACGTTAGAC ACTTAAAAGA CACTAAAGAC ATGATTACTT TAGAGGTCGT TGAAAAGGGA
721 CACTCCTCTC ATGATGAACA AACTGCTATC AAAAACAAAC TAAATATATC CAAATCTTTG
781 CACTTGGATT CTCAAGCCAA GTACTGTTTG TTAGCATTGG GCTTAGCAGA CGTATATTTA
841 CGTCTGCCTA TCAAACTTTC TTACCAAGAA AAGATCTGGG ACCATGCTGC AGGCAACGTT
901 ATTGTCCATG AAGCTGGAGG TATCCATACA GATGCCATGG AAGATGTTCC TCTAGACTTC
961 GGTAACGGTA GAACGCTAGC TACGAAGGGA GTTATAGCGT CAAGTGGCCC ACGCGAGTTA
1021 CATGACTTGG TGGTGTCTAC ATCATGCGAT GTCATTCAGT CAAGAAACGC CTAA
<210> 2
<211> 1074
<212> DNA
<213> 酵母菌3’,5’二磷酸核苷酸酶
<400> 2
1 ATGGCATTGG AAAGAGAATT ATTGGTTGCA ACTCAAGCTG TACGAAAGGC GTCTTTATTG
61 ACTAAGAGAA TTCAATCTGA AGTGATTTCT CACAAGGACT CCACTACTAT TACCAAGAAT
121 GATAATTCTC CAGTAACCAC AGGTGATTAT GCTGCACAAA CGATCATCAT AAATGCTATC
181 AAGAGCAATT TTCCTGATGA TAAGGTAGTT GGTGAAGAAT CCTCATCAGG ATTGAGCGAC
241 GCATTCGTCT CAGGAATTTT AAACGAAATA AAAGCCAATG ACGAAGTTTA TAACAAGAAT
301 TATAAAAAGG ATGATTTTCT GTTTACAAAC GATCAGTTTC CGCTAAAATC TTTGGAGGAC
361 GTCAGGCAAA TCATCGATTT CGGCAATTAC GAAGGTGGTA GAAAAGGAAG ATTTTGGTGT
421 TTGGATCCTA TTGACGGAAC CAAGGGGTTT TTAAGAGGTG AACAGTTTGC AGTATGTCTG
481 GCCTTAATTG TGGACGGTGT TGTTCAGCTT GGTTGTATTG GATGCCCCAA CTTAGTTTTA
541 AGTTCTTATG GGGCCCAAGA TTTGAAAGGC CATGAGTCAT TTGGTTATAT CTTTCGTGCT
601 GTTAGAGGTT TAGGTGCCTT CTATTCTCCA TCTTCAGATG CAGAGTCATG GACCAAAATC
661 CACGTTAGAC ACTTAAAAGA CACTAAAGAC ATGATTACTT TAGAGGCAGT TGAAAAGGGA
721 CACTCCTCTC ATGATGAACA AACTGCTATC AAAAACAAAC TAAATATATC CAAATCTTTG
781 CACTTGGATT CTCAAGCCAA GTACTGTTTG TTAGCATTGG GCTTAGCAGA CGTATATTTA
841 CGTCTGCCTA TCAAACTTTC TTACCAAGAA AAGATCTGGG ACCATGCTGC AGGCAACGTT
901 ATTGTCCATG AAGCTGGAGG TATCCATACA GATGCCATGG AAGATGTTCC TCTAGACTTC
961 GGTAACGGTA GAACGCTAGC TACGAAGGGA GTTATAGCGT CAAGTGGCCC ACGCGAGTTA
1021 CATGACTTGG TGGTGTCTAC ATCATGCGAT GTCATTCAGT CAAGAAACGC CTAA
<210> 3
<211> 1074
<212> DNA
<213> 酵母菌3’,5’二磷酸核苷酸酶
<400> 3
1 ATGGCATTGG AAAGAGAATT ATTGGTTGCA ACTCAAGCTG TACGAAAGGC GTCTTTATTG
61 ACTAAGAGAA TTCAATCTGA AGTGATTTCT CACAAGGACT CCACTACTAT TACCAAGAAT
121 GATAATTCTC CAGTAACCAC AGGTGATTAT GCTGCACAAA CGATCATCAT AAATGCTATC
181 AAGAGCAATT TTCCTGATGA TAAGGTAGTT GGTGAAGAAT CCTCATCAGG ATTGAGCGAC
241 GCATTCGTCT CAGGAATTTT AAACGAAATA AAAGCCAATG ACGAAGTTTA TAACAAGAAT
301 TATAAAAAGG ATGATTTTCT GTTTACAAAC GATCAGTTTC CGCTAAAATC TTTGGAGGAC
361 GTCAGGCAAA TCATCGATTT CGGCAATTAC GAAGGTGGTA GAAAAGGAAG ATTTTGGTGT
421 TTGGATCCTA TTGACGGAAC CAAGGGGTTT TTAAGAGGTG AACAGTTTGC AGTATGTCTG
481 GCCTTAATTG TGGACGGTGT TGTTCAGCTT GGTTGTATTG GATGCCCCAA CTTAGTTTTA
541 AGTTCTTATG GGGCCCAAGA TTTGAAAGGC CATGAGTCAT TTGGTTATAT CTTTCGTGCT
601 GTTAGAGGTT TAGGTGCCTT CTATTCTCCA TCTTCAGATG CAGAGTCATG GACCAAAATC
661 CACGTTAGAC ACTTAAAAGA CACTAAAGAC ATGATTACTT TAGAGAGCGT TGAAAAGGGA
721 CACTCCTCTC ATGATGAACA AACTGCTATC AAAAACAAAC TAAATATATC CAAATCTTTG
781 CACTTGGATT CTCAAGCCAA GTACTGTTTG TTAGCATTGG GCTTAGCAGA CGTATATTTA
841 CGTCTGCCTA TCAAACTTTC TTACCAAGAA AAGATCTGGG ACCATGCTGC AGGCAACGTT
901 ATTGTCCATG AAGCTGGAGG TATCCATACA GATGCCATGG AAGATGTTCC TCTAGACTTC
961 GGTAACGGTA GAACGCTAGC TACGAAGGGA GTTATAGCGT CAAGTGGCCC ACGCGAGTTA
1021 CATGACTTGG TGGTGTCTAC ATCATGCGAT GTCATTCAGT CAAGAAACGC CTAA
<210> 4
<211> 1074
<212> DNA
<213> 酵母菌3’,5’二磷酸核苷酸酶
<400> 4
1 ATGGCATTGG AAAGAGAATT ATTGGTTGCA ACTCAAGCTG TACGAAAGGC GTCTTTATTG
61 ACTAAGAGAA TTCAATCTGA AGTGATTTCT CACAAGGACT CCACTACTAT TACCAAGAAT
121 GATAATTCTC CAGTAACCAC AGGTGATTAT GCTGCACAAA CGATCATCAT AAATGCTATC
181 AAGAGCAATT TTCCTGATGA TAAGGTAGTT GGTGAAGAAT CCTCATCAGG ATTGAGCGAC
241 GCATTCGTCT CAGGAATTTT AAACGAAATA AAAGCCAATG ACGAAGTTTA TAACAAGAAT
301 TATAAAAAGG ATGATTTTCT GTTTACAAAC GATCAGTTTC CGCTAAAATC TTTGGAGGAC
361 GTCAGGCAAA TCATCGATTT CGGCAATTAC GAAGGTGGTA GAAAAGGAAG ATTTTGGTGT
421 TTGGATCCTA TTGACGGAAC CAAGGGGTTT TTAAGAGGTG AACAGTTTGC AGTATGTCTG
481 GCCTTAATTG TGGACGGTGT TGTTCAGCTT GGTTGTATTG GATGCCCCAA CTTAGTTTTA
541 AGTTCTTATG GGGCCCAAGA TTTGAAAGGC CATGAGTCAT TTGGTTATAT CTTTCGTGCT
601 GTTAGAGGTT TAGGTGCCTT CTATTCTCCA TCTTCAGATG CAGAGTCATG GACCAAAATC
661 CACGTTAGAC ACTTAAAAGA CACTAAAGAC ATGATTACTT TAGAGTGCGT TGAAAAGGGA
721 CACTCCTCTC ATGATGAACA AACTGCTATC AAAAACAAAC TAAATATATC CAAATCTTTG
781 CACTTGGATT CTCAAGCCAA GTACTGTTTG TTAGCATTGG GCTTAGCAGA CGTATATTTA
841 CGTCTGCCTA TCAAACTTTC TTACCAAGAA AAGATCTGGG ACCATGCTGC AGGCAACGTT
901 ATTGTCCATG AAGCTGGAGG TATCCATACA GATGCCATGG AAGATGTTCC TCTAGACTTC
961 GGTAACGGTA GAACGCTAGC TACGAAGGGA GTTATAGCGT CAAGTGGCCC ACGCGAGTTA
1021 CATGACTTGG TGGTGTCTAC ATCATGCGAT GTCATTCAGT CAAGAAACGC CTAA
<210> 5
<211> 357
<212> 氨基酸
<213> 酵母菌3’,5’二磷酸核苷酸酶
<400> 5
1 MALERELLVA TQAVRKASLL TKRIQSEVIS HKDSTTITKN DNSPVTTGDY AAQTIIINAI
61 KSNFPDDKVV GEESSSGLSD AFVSGILNEI KANDEVYNKN YKKDDFLFTN DQFPLKSLED
121 VRQIIDFGNY EGGRKGRFWC LDPIDGTKGF LRGEQFAVCL ALIVDGVVQL GCIGCPNLVL
181 SSYGAQDLKG HESFGYIFRA VRGLGAFYSP SSDAESWTKI HVRHLKDTKD MITLEVVEKG
241 HSSHDEQTAI KNKLNISKSL HLDSQAKYCL LALGLADVYL RLPIKLSYQE KIWDHAAGNV
301 IVHEAGGIHT DAMEDVPLDF GNGRTLATKG VIASSGPREL HDLVVSTSCD VIQSRNA
<210> 6
<211> 357
<212> 氨基酸
<213> 酵母菌3’,5’二磷酸核苷酸酶
<400> 6
1 MALERELLVA TQAVRKASLL TKRIQSEVIS HKDSTTITKN DNSPVTTGDY AAQTIIINAI
61 KSNFPDDKVV GEESSSGLSD AFVSGILNEI KANDEVYNKN YKKDDFLFTN DQFPLKSLED
121 VRQIIDFGNY EGGRKGRFWC LDPIDGTKGF LRGEQFAVCL ALIVDGVVQL GCIGCPNLVL
181 SSYGAQDLKG HESFGYIFRA VRGLGAFYSP SSDAESWTKI HVRHLKDTKD MITLEAVEKG
241 HSSHDEQTAI KNKLNISKSL HLDSQAKYCL LALGLADVYL RLPIKLSYQE KIWDHAAGNV
301 IVHEAGGIHT DAMEDVPLDF GNGRTLATKG VIASSGPREL HDLVVSTSCD VIQSRNA
<210> 7
<211> 357
<212> 氨基酸
<213> 酵母菌3’,5’二磷酸核苷酸酶
<400> 7
1 MALERELLVA TQAVRKASLL TKRIQSEVIS HKDSTTITKN DNSPVTTGDY AAQTIIINAI
61 KSNFPDDKVV GEESSSGLSD AFVSGILNEI KANDEVYNKN YKKDDFLFTN DQFPLKSLED
121 VRQIIDFGNY EGGRKGRFWC LDPIDGTKGF LRGEQFAVCL ALIVDGVVQL GCIGCPNLVL
181 SSYGAQDLKG HESFGYIFRA VRGLGAFYSP SSDAESWTKI HVRHLKDTKD MITLESVEKG
241 HSSHDEQTAI KNKLNISKSL HLDSQAKYCL LALGLADVYL RLPIKLSYQE KIWDHAAGNV
301 IVHEAGGIHT DAMEDVPLDF GNGRTLATKG VIASSGPREL HDLVVSTSCD VIQSRNA
<210> 8
<211> 357
<212> 氨基酸
<213> 酵母菌3’,5’二磷酸核苷酸酶
<400> 8
1 MALERELLVA TQAVRKASLL TKRIQSEVIS HKDSTTITKN DNSPVTTGDY AAQTIIINAI
61 KSNFPDDKVV GEESSSGLSD AFVSGILNEI KANDEVYNKN YKKDDFLFTN DQFPLKSLED
121 VRQIIDFGNY EGGRKGRFWC LDPIDGTKGF LRGEQFAVCL ALIVDGVVQL GCIGCPNLVL
181 SSYGAQDLKG HESFGYIFRA VRGLGAFYSP SSDAESWTKI HVRHLKDTKD MITLECVEKG
241 HSSHDEQTAI KNKLNISKSL HLDSQAKYCL LALGLADVYL RLPIKLSYQE KIWDHAAGNV
301 IVHEAGGIHT DAMEDVPLDF GNGRTLATKG VIASSGPREL HDLVVSTSCD VIQSRNA
Claims (4)
1.3’,5’-二磷酸腺苷酸特异性3’-核苷酸酶,其为以下(a)或(b)的蛋白质:
(a)由SEQ ID:NO.5所示的氨基酸序列组成的蛋白质;
(b)氨基酸序列为SEQ ID:NO.6 、SEQ ID:NO.7或SEQ ID:NO.8所示。
2.编码权利要求1所述的蛋白质的基因,该基因的核苷酸序列为SEQ ID:NO.1、SEQ ID:NO.2 、SEQ ID:NO.3或SEQ ID:NO.4所示的DNA分子。
3.一种构建权利要求1所述的3’,5’-二磷酸腺苷酸特异性3’-核苷酸酶的方法,其特征在于该方法包括以下的步骤:
1)PCR法克隆的YND核苷酸序列和pET24a原核蛋白超表达载体,分别经限制性内切酶Nde I/Hind III双酶切后,利用T4连接酶连接构建表达载体pYND,用于表达YND;
2)利用PCR突变的方法获得DNA:根据YND基因的序列与所要突变的位点,设计定点突变PCR引物,以突变位点为中心,左右各取10-15个碱基,引物长度为25-45个碱基;
3)所获DNA转化E. coli DH5α 感受态细胞扩增质粒并测序验证;
4)将突变质粒转化E. coli BL21,接种过夜培养的菌液于液体培养基中培养,收集菌体;
5)取裂解液,悬浮菌体,混合均匀后,冰浴超声波破碎菌体,离心收集上清;
6)利用PAP琼脂糖凝胶纯化YND及其突变蛋白;
所述的突变PCR引物的序列如下:
引物名称 引物序列(5’-3’)
G236A1:CATGATTACTTTAGAGGCAGTTGAAAAGGGACACTC
G236A2:GAGTGTCCCTTTTCAACTGCCTCTAAAGTAATCATG
G236C1:CATGATTACTTTAGAGTGCGTTGAAAAGGGACACTC
G236C2:GAGTGTCCCTTTTCAACGCACTCTAAAGTAATCATG
G236V1:CATGATTACTTTAGAGGTCGTTGAAAAGGGACACTC
G236V2:GAGTGTCCCTTTTCAACGACCTCTAAAGTAATCATG
G236S1:CATGATTACTTTAGAGAGCGTTGAAAAGGGACACTC
G236S2:GAGTGTCCCTTTTCAACGCTCTCTAAAGTAATCATG。
4.权利要求1所述特异性3’-核苷酸酶的应用,其特征在于:所述的特异性3’-核苷酸酶用于3’,5’二磷酸腺苷的检测。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201310339369.0A CN103451165B (zh) | 2013-08-06 | 2013-08-06 | 3’,5’-二磷酸腺苷酸特异性3’-核苷酸酶及其构建方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201310339369.0A CN103451165B (zh) | 2013-08-06 | 2013-08-06 | 3’,5’-二磷酸腺苷酸特异性3’-核苷酸酶及其构建方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN103451165A CN103451165A (zh) | 2013-12-18 |
CN103451165B true CN103451165B (zh) | 2015-08-05 |
Family
ID=49734005
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201310339369.0A Active CN103451165B (zh) | 2013-08-06 | 2013-08-06 | 3’,5’-二磷酸腺苷酸特异性3’-核苷酸酶及其构建方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN103451165B (zh) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109207485B (zh) * | 2018-09-22 | 2020-08-25 | 华中农业大学 | OsAPS1基因在改良水稻抗病性中的应用 |
CN110967451A (zh) * | 2019-12-23 | 2020-04-07 | 农业农村部环境保护科研监测所 | 一种利用离体稻穗快速鉴定水稻镉积累的方法 |
CN110982864A (zh) * | 2019-12-30 | 2020-04-10 | 孙军亭 | 一种3’,5’-二磷酸腺苷(pap)的酶合成方法及其应用 |
CN113046403B (zh) * | 2020-09-30 | 2023-04-28 | 江南大学 | 一种基于构建atp再生系统高效催化合成paps的方法 |
CN113046402B (zh) * | 2020-09-30 | 2023-04-28 | 江南大学 | 一种基于构建双功能酶合成paps的方法 |
CN113897342A (zh) * | 2021-09-22 | 2022-01-07 | 天津科技大学 | 基于分子对接的pap的高特异性的偶联酶ynd突变体、方法和应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0832970A1 (en) * | 1995-05-25 | 1998-04-01 | Ajinomoto Co., Inc. | Process for producing nucleoside-5'-phosphate |
CN101629169A (zh) * | 2009-08-06 | 2010-01-20 | 杭州利安生物科技有限公司 | 分子改造的嘌呤核苷磷酸化酶及其制备方法 |
-
2013
- 2013-08-06 CN CN201310339369.0A patent/CN103451165B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0832970A1 (en) * | 1995-05-25 | 1998-04-01 | Ajinomoto Co., Inc. | Process for producing nucleoside-5'-phosphate |
CN101629169A (zh) * | 2009-08-06 | 2010-01-20 | 杭州利安生物科技有限公司 | 分子改造的嘌呤核苷磷酸化酶及其制备方法 |
Non-Patent Citations (2)
Title |
---|
5"-腺苷磷酰硫酸激酶及其R68K 突变体对5"-腺苷-磷酸3" 羟基的磷酸化;杨洋 等;《山西农业科学》;20130630;第41卷(第6期);摘要 * |
ACCESSION:NM_001183319.1;Dujon,B.et al.;《GenBank》;20130225 * |
Also Published As
Publication number | Publication date |
---|---|
CN103451165A (zh) | 2013-12-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN103451165B (zh) | 3’,5’-二磷酸腺苷酸特异性3’-核苷酸酶及其构建方法和应用 | |
Thorson et al. | Studies of the biosynthesis of 3, 6-dideoxyhexoses: molecular cloning and characterization of the asc (ascarylose) region from Yersinia pseudotuberculosis serogroup VA | |
JP4505011B2 (ja) | 3’−ホスホアデノシン−5’−ホスホ硫酸の酵素合成法 | |
JPS61254856A (ja) | ポリヌクレオチド置換、分離、酵素開裂、およびアデノシンリン酸検出を用いる診断用試薬、キットおよび方法 | |
Zou et al. | Efficient production of deoxynucleoside-5′-monophosphates using deoxynucleoside kinase coupled with a GTP-regeneration system | |
KR100327272B1 (ko) | 뉴클레오시드5'-트리포스페이트의제조법및그응용 | |
CN110468114A (zh) | 一种聚磷酸激酶RmPPK及其编码基因和应用 | |
Zou et al. | One-pot three-enzyme synthesis of UDP-Glc, UDP-Gal, and their derivatives | |
US6040158A (en) | Process for preparing sugar nucleotide | |
Loan et al. | Recombinant cell-lysate-catalysed synthesis of uridine-5′-triphosphate from nucleobase and ribose, and without addition of ATP | |
JP3764755B2 (ja) | 「アデノシン5’−三リン酸の製造法及びその応用」 | |
JP4437786B2 (ja) | ペントース−5−リン酸エステルの製造方法。 | |
CN104946674B (zh) | 一种腺苷三磷酸/双磷酸酶及其编码基因与应用 | |
JP3545425B2 (ja) | ウリジン二リン酸―n―アセチルグルコサミンの製造法 | |
Mizanur et al. | Cloning and characterization of a heat-stable CMP-N-acylneuraminic acid synthetase from Clostridium thermocellum | |
Taran et al. | Synthesis of 2-chloro-2′-deoxyadenosine by microbiological transglycosylation using a recombinant Escherichia coli strain | |
Kang et al. | Preparative synthesis of dTDP‐l‐rhamnose through combined enzymatic pathways | |
Ding et al. | Enzymatic synthesis of nucleosides by nucleoside phosphorylase co-expressed in Escherichia coli | |
Fang et al. | A chemoenzymatic route to synthesize unnatural sugar nucleotides using a novel N-acetylglucosamine-1-phosphate pyrophosphorylase from Camphylobacter jejuni NCTC 11168 | |
Yan et al. | Characterization of a family I-liked alkaline-resistant inorganic pyrophosphatase from the hyperthermophilic archaeon Pyrococcus furiosus for rapid determination of sugar nucleotidyltransferase at high temperature | |
CN108795919B (zh) | 一种催化制备udp-鼠李糖的融合酶及其应用 | |
Gao et al. | Substrate promiscuity of pyruvate kinase on various deoxynucleoside diphosphates for synthesis of deoxynucleoside triphosphates | |
WO2021031170A1 (zh) | 一种聚磷酸激酶RmPPK及其编码基因和应用 | |
Diricks | Unlocking nature’s glycosylation potential: characterization and engineering of novel sucrose/trehalose synthases | |
JP2002335988A (ja) | オリゴ糖の製造法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
TR01 | Transfer of patent right |
Effective date of registration: 20191225 Address after: 314416 No.318, Nanjie Road, Yuanhua Town, Haining City, Jiaxing City, Zhejiang Province Patentee after: Haining Yuanhua Town Industrial Investment Co., Ltd Address before: 321004 No. 688 Yingbin Road, Zhejiang, Jinhua Patentee before: Zhejiang Normal University |
|
TR01 | Transfer of patent right |