CN102154190A - Engineering escherichia coli capable of efficiently producing hyaluronic acid and preparation method thereof - Google Patents
Engineering escherichia coli capable of efficiently producing hyaluronic acid and preparation method thereof Download PDFInfo
- Publication number
- CN102154190A CN102154190A CN 201110006213 CN201110006213A CN102154190A CN 102154190 A CN102154190 A CN 102154190A CN 201110006213 CN201110006213 CN 201110006213 CN 201110006213 A CN201110006213 A CN 201110006213A CN 102154190 A CN102154190 A CN 102154190A
- Authority
- CN
- China
- Prior art keywords
- gene
- seq
- site
- sequence table
- primer
- 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.)
- Granted
Links
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 40
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 35
- 229960003160 hyaluronic acid Drugs 0.000 title claims abstract description 34
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 29
- 239000013604 expression vector Substances 0.000 claims abstract description 21
- 102000003960 Ligases Human genes 0.000 claims abstract description 4
- 108090000364 Ligases Proteins 0.000 claims abstract description 4
- 241000894006 Bacteria Species 0.000 claims description 57
- 230000000968 intestinal effect Effects 0.000 claims description 32
- 108020004414 DNA Proteins 0.000 claims description 29
- 108090000320 Hyaluronan Synthases Proteins 0.000 claims description 28
- 230000001580 bacterial effect Effects 0.000 claims description 24
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 22
- 210000001072 colon Anatomy 0.000 claims description 21
- 239000012634 fragment Substances 0.000 claims description 21
- 101710088194 Dehydrogenase Proteins 0.000 claims description 20
- 101150105185 kfiD gene Proteins 0.000 claims description 20
- 239000013612 plasmid Substances 0.000 claims description 18
- 241000606856 Pasteurella multocida Species 0.000 claims description 17
- 238000004519 manufacturing process Methods 0.000 claims description 17
- 229940051027 pasteurella multocida Drugs 0.000 claims description 17
- 238000011144 upstream manufacturing Methods 0.000 claims description 16
- HSCJRCZFDFQWRP-RDKQLNKOSA-N UDP-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-RDKQLNKOSA-N 0.000 claims description 15
- 239000013598 vector Substances 0.000 claims description 15
- 238000012408 PCR amplification Methods 0.000 claims description 12
- 230000004087 circulation Effects 0.000 claims description 12
- 238000013461 design Methods 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 230000029087 digestion Effects 0.000 claims description 10
- 101150097493 D gene Proteins 0.000 claims description 9
- 230000003321 amplification Effects 0.000 claims description 9
- 230000008859 change Effects 0.000 claims description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 9
- 108091008146 restriction endonucleases Proteins 0.000 claims description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 7
- 241000178955 Pasteurella multocida subsp. multocida Species 0.000 claims description 7
- 238000004458 analytical method Methods 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- MVMSCBBUIHUTGJ-LRJDVEEWSA-N GDP-alpha-D-glucose Chemical compound C([C@H]1O[C@H]([C@@H]([C@@H]1O)O)N1C=2N=C(NC(=O)C=2N=C1)N)OP(O)(=O)OP(O)(=O)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O MVMSCBBUIHUTGJ-LRJDVEEWSA-N 0.000 claims description 6
- 108091081024 Start codon Proteins 0.000 claims description 6
- 108020005038 Terminator Codon Proteins 0.000 claims description 6
- 230000004186 co-expression Effects 0.000 claims description 6
- 238000001228 spectrum Methods 0.000 claims description 6
- XUWPJKDMEZSVTP-LTYMHZPRSA-N kalafungina Chemical compound O=C1C2=C(O)C=CC=C2C(=O)C2=C1[C@@H](C)O[C@H]1[C@@H]2OC(=O)C1 XUWPJKDMEZSVTP-LTYMHZPRSA-N 0.000 claims description 5
- 101710128038 Hyaluronan synthase Proteins 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 238000001712 DNA sequencing Methods 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 230000010076 replication Effects 0.000 claims description 3
- 238000012772 sequence design Methods 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 claims description 3
- 239000002775 capsule Substances 0.000 claims description 2
- 238000003259 recombinant expression Methods 0.000 abstract description 3
- 229910019142 PO4 Inorganic materials 0.000 abstract description 2
- HSCJRCZFDFQWRP-JZMIEXBBSA-N UDP-alpha-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-JZMIEXBBSA-N 0.000 abstract description 2
- HSCJRCZFDFQWRP-UHFFFAOYSA-N Uridindiphosphoglukose Natural products OC1C(O)C(O)C(CO)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-UHFFFAOYSA-N 0.000 abstract description 2
- 239000010452 phosphate Substances 0.000 abstract 1
- 230000014509 gene expression Effects 0.000 description 22
- 239000000047 product Substances 0.000 description 22
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- 230000000694 effects Effects 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 102000018866 Hyaluronan Receptors Human genes 0.000 description 9
- 108010013214 Hyaluronan Receptors Proteins 0.000 description 9
- 241000196324 Embryophyta Species 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 230000002068 genetic effect Effects 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 102000003918 Hyaluronan Synthases Human genes 0.000 description 6
- 150000002460 imidazoles Chemical class 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 239000001488 sodium phosphate Substances 0.000 description 6
- 235000011008 sodium phosphates Nutrition 0.000 description 6
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical class [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 108700005078 Synthetic Genes Proteins 0.000 description 4
- XCCTYIAWTASOJW-XVFCMESISA-N Uridine-5'-Diphosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 XCCTYIAWTASOJW-XVFCMESISA-N 0.000 description 4
- 239000000287 crude extract Substances 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
- 238000011160 research Methods 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000012269 metabolic engineering Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 230000009261 transgenic effect Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- XQSPNYNSJBNQHQ-DBKUKYHUSA-N C(C)(=O)C1(O)[C@H](N)[C@@H](O)[C@H](O)[C@H](O1)CO.[N] Chemical compound C(C)(=O)C1(O)[C@H](N)[C@@H](O)[C@H](O)[C@H](O1)CO.[N] XQSPNYNSJBNQHQ-DBKUKYHUSA-N 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 206010018325 Congenital glaucomas Diseases 0.000 description 2
- 206010012565 Developmental glaucoma Diseases 0.000 description 2
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 2
- 208000007157 Hydrophthalmos Diseases 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 241000701245 Paramecium bursaria Chlorella virus 1 Species 0.000 description 2
- 230000010748 Photoabsorption Effects 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 201000001024 buphthalmos Diseases 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 229920002301 cellulose acetate Polymers 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 229940097043 glucuronic acid Drugs 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- SHFJWMWCIHQNCP-UHFFFAOYSA-M hydron;tetrabutylazanium;sulfate Chemical compound OS([O-])(=O)=O.CCCC[N+](CCCC)(CCCC)CCCC SHFJWMWCIHQNCP-UHFFFAOYSA-M 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 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
- LZDKZFUFMNSQCJ-UHFFFAOYSA-N 1,2-diethoxyethane Chemical compound CCOCCOCC LZDKZFUFMNSQCJ-UHFFFAOYSA-N 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000561734 Celosia cristata Species 0.000 description 1
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 1
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 description 1
- 241001646716 Escherichia coli K-12 Species 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010028921 Lipopeptides Proteins 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 description 1
- 241000223785 Paramecium Species 0.000 description 1
- 241000606860 Pasteurella Species 0.000 description 1
- XCCTYIAWTASOJW-UHFFFAOYSA-N UDP-Glc Natural products OC1C(O)C(COP(O)(=O)OP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 XCCTYIAWTASOJW-UHFFFAOYSA-N 0.000 description 1
- 102000057144 Uridine Diphosphate Glucose Dehydrogenase Human genes 0.000 description 1
- 108010054269 Uridine Diphosphate Glucose Dehydrogenase Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000000516 activation analysis Methods 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 1
- AEMOLEFTQBMNLQ-WAXACMCWSA-N alpha-D-glucuronic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-WAXACMCWSA-N 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000000118 anti-neoplastic effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 208000027697 autoimmune lymphoproliferative syndrome due to CTLA4 haploinsuffiency Diseases 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000001520 comb Anatomy 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 229920006037 cross link polymer Polymers 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 230000008175 fetal development Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- KIUKXJAPPMFGSW-MNSSHETKSA-N hyaluronan Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H](C(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-MNSSHETKSA-N 0.000 description 1
- 229940099552 hyaluronan Drugs 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 238000005461 lubrication Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- MBLBDJOUHNCFQT-UHFFFAOYSA-N n-(3,4,5,6-tetrahydroxy-1-oxohexan-2-yl)acetamide Chemical compound CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000000710 polymer precipitation Methods 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000000250 revascularization Effects 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention provides an engineering escherichia coli capable of efficiently producing hyaluronic acid (HA) and a preparation method thereof. The engineering escherichia coli is obtained by transferring escherichia coli through constructing recombinant expression vector commonly expressed in escherichia coli by gene Has containing HA synthetase and gene containing uridine diphosphate glucose phosphate dehydrogenate. The engineering escherichia coli capable of efficiently producing HA, provided by the invention, is pHK/JM109 with CGMCC No.3926. The engineering escherichia coli provided by the invention can produce 29-2.5g/L of HA. Compared with the yield in the prior art, the highest yield of HA obtained from gene spHas synthesized by Gram positive strep HA which is expressed in escherichia coli is enhanced by about more than 10 times.
Description
Technical field
The present invention relates to the gene engineering technology field of microorganism, specifically is efficient product hyaluronic acid engineering colon bacillus that makes up with the plasmid form or in the genome conformity mode and preparation method thereof.
Background technology
E. coli k-12 strain system because of genetic background clearly, have numerous genetic transformation systems efficiently and genomic ease for operation is widely used in the metabolic engineering field, to produce high value added product miscellaneous.Do not find as yet at present that natural coli strain has the hyaluronic ability of production.
(Hyaluronic acid HA) separates obtain a kind of and has the high viscosity glycosaminoglycan hyaluronic acid the earliest from buphthalmos.Hyaluronic acid extensively exists in the humans and animals tissue, and its molecular weight is 5 * 10
4To 8 * 10
6Between the dalton, be by β-1, glucuronic acid and β-1 that the 4-glycosidic link connects, nitrogen acetylglucosamine (the β 1-4 D-Glucuronic acid that the 3-glycosidic link connects, GlcA, β 1-3 D-N-acetylglucosamine, the straight-chain polymer that GlcNAc) alternately is formed by connecting.Studies confirm that this polysaccharide has participated in numerous physiological activities such as mammiferous fertilization, fetal development, revascularization and joint lubrication as structure and signaling molecule; Also playing an important role aspect inflammation and injury repairing, the moisture of keeping skin and the elasticity simultaneously.
Discover that the HA oligonucleotide chain of length-specific is being brought into play important function at aspects such as the unusual reparations of antineoplastic immune.Because hyaluronic acid unique chemical characteristic and physiological function, HA and the oligonucleotide chain that comes from HA are widely used in all many-sides such as ophthalmologic operation, arthritis treatment, antitumor, administering mode, cosmetic industry, only HA in 2008 is as raw material, and the dealing money in the world market is about 1,000,000,000 dollars and the trend that increases is year by year arranged.
Occurring in nature HA extensively exists, as the cell envelope of animal tissues, Mammals cause of disease bacterium and the chlorella of viral PBCV-1 dip-dye etc.Present commercial HA mainly from the pathogenic bacteria of animal tissues's (as cockscomb, umbilical cord, buphthalmos, knuckle synovia) and cultivator and higher mammal (as strep,
Streptococci) the middle extraction.For the consideration of aspects such as resource limit, cost problem and safety of medicine, above-mentioned production is ideal style absolutely not, and the method for more economic, safe and lasting production HA is done one's utmost to explore by international community.
Molecular biology and development of biology are for the solution of the problems referred to above provides important means.To HA synthesis mechanism in animal and microorganism, the researchs such as clone of the building-up process indispensable gene of many HA all are the means by metabolic engineering, and reconstruction or transformation route of synthesis are produced HA and established solid basis in the host living beings of safety.
Japanese scholar in 2004
Chai paddy grows youth etc.(Chinese patent application number be 200480022328.8) attempts HA synthesizing in plant tissue by express the HA synthetic gene (Has) that obtains from human and paramecium virus (PBCV-1) in transgenic plant.Widner B etc. (Bill Widner et al., Hyaluronic Acid Production in
Bacillus subtilis Appl. Environ. Microbiol. 2005,71 (7): 3747 – 3752) successful expression obtains from gram-positive strep in subtilis HA synthetic gene, the engineering genus bacillus is fermented in common triangular flask and obtains 0.5-0.6g/L HA content.
Yet produce hyaluronic acid in plant, because of the conversion difficulty of plant, growth cycle is subject to seasonal restrictions, and many conservative environmentalists oppose transgenic plant, and there is environmentally safe potential hazard etc. in transgenic plant itself and is subjected to application limitations.For subtilis, because of this bacterium has the generation intracellular toxin, the report of various big ring grease classes and lipopeptide antibiotic, and can cause through fermentation, be used for the separation and purification cost up of field of medicaments HA, serial problems such as productive rate reduction.
Many intestinal bacteria that derive from human intestinal are beneficial bacterias of safeguarding intestinal microbial balance and HUMAN HEALTH.In addition e. coli k12 strain cording clearly genetic background, have numerous genetic transformation systems efficiently and genome operability, and be widely used in the biological product of metabolic engineering field with the production high added value.For being that the host carries out the Stephanopoulos G study group that the hyaluronic reported first of engineered production is a Massachusetts Institute Technology with intestinal bacteria.2008 these study group pass through at expression in escherichia coli Gram-positive strep HA synthetic gene
SpHasWith brand-new synthetic, optimized codon and be used for escherichia coli expression
SeHasGene, this research obtain the HA output of the highest about 0.2g/L.This project intestinal bacteria are difficult to obtain the industrialization utilization because of the HA synthetic yield is low.
Summary of the invention
The purpose of this invention is to provide a kind of efficient product hyaluronic acid engineering colon bacillus and preparation method thereof, overcome and produce hyaluronic shortage of resources, safety issue and low hyaluronic acid synthetic problem in engineering colon bacillus in the prior art.
The present invention efficiently produces the hyaluronic acid engineering colon bacillus with expressing hyaluronan synthase gene (hyaluronic acid synthase Has) and uridine diphosphoglucose dehydrogenase gene (UDP-glucose Dehydrogenase).
The efficient product hyaluronic acid engineering colon bacillus that contains hyaluronan synthase gene and uridine diphosphoglucose dehydrogenase gene of the present invention is to make up acquisition by the following method:
(1) carrier of construction expression hyaluronan synthase gene Has specifically is that the Has that PCR clones is connected in the colibacillary expression vector (as pQE80l);
(2) carrier of construction expression uridine diphosphoglucose dehydrogenase gene, the uridine diphosphoglucose dehydrogenase gene that PCR is cloned connects in the colibacillary expression vector (as pQE80l);
(3) structure contains hyaluronan synthase gene Has
,With contain the uridine diphosphoglucose dehydrogenase gene, the recombinant vectors of co expression in intestinal bacteria; The uridine diphosphoglucose dehydrogenase gene expression carrier that contains with structure in (2) is the template of PCR, and amplification contains driven element (as T5) and drives
KfiThe PCR product that D expresses, and be connected into the expression hyaluronan synthase gene that makes up in (1)
HasExpression vector in and obtain to contain hyaluronan synthase gene
HasWith the uridine diphosphoglucose dehydrogenase gene, and can be in intestinal bacteria the recombinant vectors of co expression.
(4) hyaluronan synthase gene will be obtained to contain in (3)
HasWith the recombinant vectors of uridine diphosphoglucose dehydrogenase gene, transformed into escherichia coli (as the JM109 bacterial strain) obtains the hyaluronic engineering colon bacillus of efficient product through screening.
Product hyaluronic acid engineering colon bacillus of the present invention is pHK/JM109, classification called after colon bacillus (
Escherichia coli), preserving number is CGMCC No.3926, China Committee for Culture Collection of Microorganisms common micro-organisms center (the being called for short CGMCC) preservation on June 17th, 2010 in the BeiJing, China city, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Efficient product hyaluronic acid engineering colon bacillus (pHK/ of the present invention
E.coliJM109) specifically preparation by the following method:
(1) make up the expression vector pQEpmHas contain hyaluronan synthase gene, with the pcr amplification method clone be derived from Pasteurella multocida (
Pasteurella multocidaSubsp.
Multocida) in hyaluronan synthase gene (
PAsteurella
mUltocida
hYaluronic
aCid
sYnthase
PmHas), the design of its pcr amplification clone the primer is as follows:
Upstream primer pmHas P1 is 5'-TA
GGATCC ATGAACACATTATCACAAGCAAT-3', (SEQ ID No:3 in the sequence table) contains
BamH I site GGATCC and initiator codon ATG;
Downstream primer is
PmHasP2 is 5'-TA
GAGCTC TTASEQ ID No:4 in the TAGAGTTATACTATTAATAAT-3'(sequence table), contain
SacI site GAGCTC and terminator codon TAA;
The masterplate of PCR is the genomic dna of Pasteurella multocida strains A TCC 15742, the amplified production warp
BamH I and
SacBehind the I double digestion, connect into the identical restriction enzyme site among the commercial carrier pQE80L, insert fragment after DNA order-checking conclusive evidence does not have sudden change, with obtaining carrier called after pQEpmHas, gene is
PmHas(SEQ ID No:1 in the sequence table)
(2) make up the expression vector pQEkfiD contain the uridine diphosphoglucose dehydrogenase gene, with the pcr amplification method clone be derived from come from intestinal bacteria (
Escherichia coli, E.coli) bacterial strain k5(
E. coliK5) GDPG dehydrogenase gene (K in
5Capsule gene D,
KfiD), the design of primers of amplification is as follows:
Upstream primer
KfiD P1:5'-TGG
AGATCT ATGSEQ ID No:5 in the TTCGGAACACTAAAAATAACT G-3'(sequence table), contain Bgl II site AGATCT and initiator codon ATG);
Downstream primer
KfiD P2:5'-TT
CTCGAG TTASEQ ID No:6 in the GTCACATTTAAA CAAATCGCGAC-3'(sequence table), contain
XhoI site CTCGAG and terminator codon TAA;
The masterplate of PCR is the genomic dna of intestinal bacteria bacterium k5 strains A TCC 23500, the also product of purifying that successfully increases, warp
BglII and
XhoBehind the I double digestion, connect into the commercial carrier pQE80L's of Qiagen company
BamH I and
SalI site, gained carrier after DNA sequencing analysis conclusive evidence is inserted fragment and is not had sudden change, the carrier called after pQEkfiD that obtains, corresponding gene called after
KfiD gene (SEQ ID No:2 in the sequence table);
(3) make up co expression hyaluronan synthase gene and the recombinant vectors that contains the uridine diphosphoglucose dehydrogenase gene in the intestinal bacteria:
According to the DNA sequences Design PCR primer of the pQEkfiD carrier that builds, amplification T5 promoters driven
KfiThe fragment T5:kfiD that D expresses, concrete primer sequence design is as follows:
Upstream primer T5kfiD P1:5'-TA
GAGCTCCCTTTCGTCTTCAC
GSEQ ID No:7 in the TCGAGA-3'(sequence table), this primer contains
SacThe I site, GAGCTC, wherein subscript G represents C in the original pQE80L carrier is sported G, to eliminate it
XhoThe l site, CTCGAG;
Downstream primer T5kfiD P2:5'-TA
CTCGAGSEQ ID No:8 in the TTCTGAGGTCATTACTGGATCT-3'(sequence table), this primer contains
XhoI site, CTCGAG;
The masterplate of PCR pQEkfiD carrier, the T5:kfiD amplified production of purifying, at first connect into the pGEM-T-Vector(Promega product, www.promega.com) and transformed into escherichia coli JM109 bacterial strain, obtain intermediate carrier pT-T5KfiD, SEQ ID No:9 in the T5:kfiD(sequence table in this carrier) fragment, after sequencing analysis confirmed that nucleotide sequence does not have sudden change, this inserted T5:kfiD fragment warp
SacI and
XhoBehind the I double digestion, connect into expression vector pQEpmHas's
SacI and
SalI site and obtaining
PmHasWith
KfiThe recombinant vectors pQHK of D gene coexpression in intestinal bacteria;
(4) structure of pHK
Dna sequence dna (GenBank No. Y14439) design primer according to the Gram-negative bacteria host wide spectrum plasmid pBBR122 of German MoBiTec company:
Upstream primer PBBR P1:5'-TTTGGT
GTCGACSEQ ID NO:10 in the CTTGCCAGCCCGTGGATATGTGG-3'(sequence table); Contain
SalThe I restriction enzyme site, GTCGAC
Downstream primer PBBR P1:5'-TTAGGT
GTCGACSEQ ID NO:11 in the TCTGTGATGGCTTCCATGTCGGCAG-3'(sequence table), contain
SalThe I restriction enzyme site, GTCGAC);
With comprising its replication site and kalamycin resistance gene among the above-mentioned primer amplification plasmid pBBR122 in interior fragment, the parameter of PCR amplification is: 95 ℃, 2min, 1 circulation; 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 3min, 25 circulations; 72 ℃, 1 circulation of 10min, the about 3.2 kb amplified productions of purifying are used
SalBehind the I enzymolysis, add ligase enzyme and connect certainly, self connect product transformed into escherichia coli JM109 bacterial strain, and obtain plasmid pRP (SEQ ID NO:12 in the sequence table), extract plasmid pRP, warp with the kantlex screening by it
SalBehind the I enzymolysis, connect into above-mentioned can be in intestinal bacteria coexpression pmHas and
KfiThe pQHK's of D gene
XhoThe I site, and obtain coexpression in Gram-negative bacteria
PmHasWith
KfiD synthesizes hyaluronic expression vector pHK;
(5) this recombinant vectors pHK is changed over to the hyaluronic engineering colon bacillus pHK/JM109(CGMCC of the described efficient product of acquisition NO:3926 in the e. coli jm109).
Above-described coli strain pHK/JM109 is the efficient hyaluronic acid engineering colon bacillus that produces, and wherein pHK is the host strain of expression vector for expression vector JM109.
Compared with prior art, the invention has the beneficial effects as follows:
1, engineering colon bacillus of the present invention can produce hyaluronic acid 2g/L~2.5g/L, than in the prior art by expression in escherichia coli Gram-positive strep HA synthetic gene spHas and brand-new synthetic, the hyaluronic acid volume of production of the highest about 0.2g/L that optimized codon and be used for that the seHas gene of escherichia coli expression obtains improves nearly 10 times.
2, the present invention is used in the hyaluronan synthase gene pmHas of derived from gram-negative bacteria for the first time and contains the uridine diphosphoglucose dehydrogenase gene
KfiD expresses in the Gram-negative host and produces hyaluronic acid.
3, the constructed recombinant expression vector pHK of the present invention contains the essential fragment that can duplicate of wide spectrum in gram negative bacterium, and drives hyaluronan synthase gene pmHas and contain the uridine diphosphoglucose dehydrogenase gene
KfiThe D expression promoter has activity preferably, and makes recombinant expression vector pHK of the present invention in most of gram negative bacterium, can be used for producing hyaluronic acid in (other non-intestinal bacteria) gram negative bacterium.
Description of drawings
Fig. 1 is that hyaluronic acid synthetase catalysis GDPG acid and the polymerization of GDPG amine form hyaluronic procedure chart among the present invention.
Fig. 2 is a coexpression of the present invention
PmHaS and
KfiDCarrier figure, wherein A:pQHK only is used at expression in escherichia coli; B:pHK both can be used for can also being used for expressing at other Gram-negative bacterias at expression in escherichia coli;
Fig. 3 be the matter acid synthase gene that shows transparency of the present invention (
PmHas) and contain the uridine diphosphoglucose dehydrogenase gene (
KfiD) the expression figure in the e. coli jm109 bacterial strain, pmHAS detection of expression in the A:pQEpmHAS/JM109 bacterial strain wherein, pmHAS and kfiD coexpression detect in the B:pHK/JM109 bacterial strain. A1, B1 is respectively pQEpmHAS/JM109 bacterial strain and pHK/JM109 bacterial strain, electrophoresis through the Ni-NTA-Agrose purifying, show that the pmHas molecular weight is about 110kD, the kfiD molecular weight is about 45kD, A2, B2 is that (molecular weight is respectively 225 150 100 75 50 35 25 15 (KDa) to the molecular weight of albumen standard from top to bottom, A3, B3: the total soluble protein electrophoresis that is respectively pQE80L/JM109 and pBQ/JM109 (empty carrier conversion), A4, B4 are respectively the total soluble protein electrophoresis of pQEpmHas/M109 and pHK/JM109.
Dna sequence dna explanation involved in the present invention
In the sequence table shown in the SEQ ID NO:1 be derived from Pasteurella multocida (
Pasteurella multocidaSubsp.
MultocidaIn hyaluronan synthase gene
PmHasThe coding region nucleotide sequence.
In the sequence table shown in the SEQ ID NO:2 is to be derived from intestinal bacteria k5 bacterial strain (middle GDPG dehydrogenase gene
KfiDThe coding region nucleotide sequence.
In the sequence table shown in the SEQ ID NO:3 be clone be derived from Pasteurella multocida (
Pasteurella multocidaSubsp.
Multocida) in hyaluronan synthase gene
PmHasThe base sequence of upstream primer.
In the sequence table shown in the SEQ ID NO:4 be clone be derived from Pasteurella multocida (
Pasteurella multocidaSubsp.
Multocida) in hyaluronan synthase gene
PmHasThe base sequence of downstream primer.
In the sequence table shown in the SEQ ID NO:5 is that the clone comes from GDPG dehydrogenase gene in the intestinal bacteria k5 bacterial strain
KfiThe base sequence of the upstream primer of D.
In the sequence table shown in the SEQ ID NO:6 is that the clone comes from GDPG dehydrogenase gene in the intestinal bacteria k5 bacterial strain
KfiThe base sequence of the downstream primer of D.
In the sequence table shown in the SEQ ID NO:7 is the base sequence of the segmental upstream primer of clone T5:kfiD.
In the sequence table shown in the SEQ ID NO:8 is the base sequence of the segmental downstream primer of clone T5:kfiD.
In the sequence table shown in the SEQ ID NO:9 is the segmental nucleotide sequence of T5:kfiD.
In the sequence table shown in the SEQ ID NO:10 is clone's PBRR122 kalamycin resistance gene and the base sequence that duplicates essential segmental upstream primer.
In the sequence table shown in the SEQ ID NO:11 is clone's PBRR122 kalamycin resistance gene and the base sequence that duplicates essential segmental downstream primer.
In the sequence table shown in the SEQ ID NO:12 is the nucleotide sequence of plasmid pRP.
The genetic resources that the present invention relates to
1: kill Bath DS strains A TCC15742(Pasteurella multocida subsp. multocida (Lehmann and Neumann) Rosenbusch and Merchant) more; Genetic resources is taken from: U.S.'s typical case's culture is preserved center (ATCC); Obtain manner: buy price 225 U.S. dollars; Acquisition time: in January, 2008; Primary source: U.S. scientist KL Heddleston separates acquisition time from the turkey heart: in April, 1962
2: intestinal bacteria K5 strains A TCC23500(
Escherichia coli(Migula) Castellani and Chalmers, serotype O2a, 2b:K5 (L): H4; Genetic resources is taken from: U.S.'s typical case's culture is preserved center (ATCC) obtain manner: buy, price 225 U.S. dollars, acquisition time: in September, 2008 primary source: the Center for Disease Control (CDC) scientist Kauffmann separates from people urine, and acquisition time: July nineteen forty-three.
Embodiment
The present invention is for a more detailed description by following examples, but the present invention is not limited to this.
Embodiment
Be derived from Pasteurella multocida (
Pasteurella multocidaSubsp.
Multocida) in hyaluronan synthase gene
PmHasThe clone and contain hyaluronan synthase gene
PmHasStructure and the transformed into escherichia coli JM109 bacterial strain of expression vector pQEpmHas.Concrete steps are as follows:
(1) make up the expression vector pQEpmHas contain hyaluronan synthase gene, with the pcr amplification method clone be derived from Pasteurella multocida (
Pasteurella multocidaSubsp.
Multocida) in hyaluronan synthase gene (
PAsteurella
mUltocida
hYaluronic
aCid synthase
PmHas), the design of its pcr amplification clone the primer is as follows:
Upstream primer pmHas P1 is 5'-TA
GGATCC ATGAACACATTATCACAAGCAAT-3', (SEQ ID No:3 in the sequence table) contains
BamH I site GGATCC and initiator codon ATG;
Downstream primer is
PmHasP2 is 5'-TA
GAGCTC TTASEQ ID No:4 in the TAGAGTTATACTATTAATAAT-3'(sequence table), contain
SacI site GAGCTC and terminator codon TAA;
The masterplate of PCR is the genomic dna of Pasteurella multocida strains A TCC 15742, the amplified production warp
BamH I and
SacBehind the I double digestion, connect into the identical restriction enzyme site among the commercial carrier pQE80L, insert fragment after DNA order-checking conclusive evidence does not have sudden change, with obtaining carrier called after pQEpmHas, gene is
PmHasShown in SEQ ID NO:1 in the sequence table.
In the intestinal bacteria
PmHasExpress purifying and determination of activity:
To contain pQEpmHas e. coli jm109 bacterial strain, containing 100ug/L Amp(acillin) LB(agar solid) substratum, 10g/L Tryptone(tryptone), 5g/L Yeast extract(yeast extract), 10g/L NaCl, pH 7.0, and line is producing single bacterium colony on the 15g/L agar plate, and picking list bacterium colony places LB that 5ml contains 100u/L Amp to cultivate and obtained seed liquor in 14-16 hour at 37 ℃, 250 rpm; Seed liquor is grown to the about 0.5-0.6 of culture 600nm photoabsorption by 100 times (V/V) cultivation in the new LB that contains 100u/L Amp; At this moment add the isopropyl-that final concentration is 1mM at once, isopropyl-beta-D-thiogalactoside (IPTG), and 30 ℃ of cultivations 6-8 hour.Culture is collected bacterium through the centrifugal 5min of 12000 rpm, thalline equal-volume 50 mM(pH8.0) after the sodium phosphate buffer washing, recentrifuge is collected, thalline is suspended in pH8.0 with about 0.2g fresh weight/ml concentration, contain 50 mM sodium phosphates, 300mM NaCl, 10mM imidazoles (imidazole), the 1mM N,O-Diacetylmuramidase, after in the mixed solution of the proteinase inhibitor of 2mM (DMSF), (0-4 ℃) ultrasonic disruption is homogenate under the low temperature, and cell lysate is got supernatant liquor and got through centrifugal 20 min of 12000 RPM
PmHasCrude extract.This crude extract joins in the chromatography column that contains Ni-NTA-Agrose (Sigma company product), with pH 8.0, contain the 50mM sodium phosphate, 300mM NaCl, 20 mM imidazoles (imidazole) solution washings and with pH 8.0, contain 50 mM sodium phosphates, 300mM NaCl, 250 mM imidazoles (imidazole) eluant solutions are collected elutriant.This elute soln is through 10mM pH7.0 Tris-HCl dialysed overnight, purifying hyaluronic acid synthetase (
PmHas), from the visible hyaluronic acid synthetase of Fig. 3 (
PmHas) in the e. coli jm109 bacterial strain, obtain to efficiently express, and be purified as the single band of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The activation analysis of hyaluronic acid synthetase adopts external hyaluronic accumulation to measure, enzymatic reaction is to carry out in the 50ul system, this system contains 100mM Tris HCl (pH7.0), 40mM MgSO4,0.5mM ethylene glycol diethyl ether tetraacethyl (EGTA), 2mM, mercaptoethanol 0.1% bovine serum albumin, (BSA), 2mM uridine diphosphate (UDP) 6-glucuronic acid (UDP-GlcA) is in 2mM uridine diphosphate (UDP) nitrogen acetylglucosamine (UDP-GlcNAc) dilution different with 10ul, the different purge process and the pmHas enzyme liquid of different steps.After reactant mixes, behind reaction 60min under 37 ℃ of conditions, make the enzyme deactivation termination reaction with 90 ℃ of 5min, centrifugal reaction mixture is got supernatant and is measured hyaluronic acid contents.
Hyaluronic acid contents is measured and is adopted hyaluronic acid binding protein (Hyaluronic Acid Binding Protein, HABP) measure, the present invention adopts hyaluronic acid radioimmunoassay kit (Beijing North biotechnology research institute product, http://www.bnibt.com,) analyze, this assay kit adopts competition radio immunoassay, promptly HA in standard or the testing sample and 125I-HA common with the hyaluronic acid binding protein (HABP) of limiting the quantity of being at war with property association reaction under suitable condition.Part 125I-HA combines with HABP and forms mixture, and another part is unbound state.125I-HA and HABP bonded ratio depend on the content of non-marked HA in standard or the testing sample, and the content of non-marked HA is high more, and the mixture that 125I-HA and HABP form is few more.HA-HABP mixture and HABP antibody and second antibody form crosslinked polymer precipitation gets off, and measures sedimentary radiocounting.Can obtain typical curve and regression equation through after the data processing, find the HA content of sample from typical curve.1 unit of enzyme activity (U) is defined as: catalysis in 1 hour forms 1ug hyaluronan molecule number.Protein content employing Bole (Bio-Rad) company's protein determination kit (www.biorad.com) is a protein standard substance with bovine serum albumin (BSA) also in the enzyme liquid, lives with the ratio that calculates enzyme.Table 1 data show
PmHasGene obtains activity expression in intestinal bacteria.
Remarks: data are the mean+/-standard error of 3 mensuration in the table 1
Upstream primer
KfiD P1:5'-TGG
AGATCT ATGSEQ ID No:5 in the TTCGGAACACTAAAAATAACT G-3'(sequence table), contain Bgl II site AGATCT and initiator codon ATG);
Downstream primer
KfiD P2:5'-TT
CTCGAG TTASEQ ID No:6 in the GTCACATTTAAA CAAATCGCGAC-3'(sequence table), contain
XhoI site CTCGAG and terminator codon TAA;
The masterplate of PCR is the genomic dna of intestinal bacteria k5 strains A TCC 23500, the also product of purifying that successfully increases, warp
BglII and
XhoBehind the I double digestion, connect into the commercial carrier pQE80L's of Qiagen company
BamH I and
SalI site, gained carrier after DNA sequencing analysis conclusive evidence is inserted fragment and is not had sudden change, the carrier called after pQEkfiD that obtains, corresponding gene called after
KfiThe D gene is shown in SEQ ID No:2 in the sequence table.
In the intestinal bacteria
KfiThe D expression of gene, purifying and determination of activity:
KfiThe expression of D, purifying is similar to the expression and purification of above-mentioned hyaluronic acid synthetase, specifically is, will contain the e. coli jm109 bacterial strain of pQE kfiD, grows single bacterium colony containing on 100ug/L acillin (Amp) the LB Agar solid medium.Single bacterium colony places 5ml to contain the LB of 100u/L Amp at 37 ℃, 250 rpm cultivated after 14-16 hour, culture is diluted among the LB by 100 times (V/V), 37 ℃ are cultured to OD600nm photoabsorption (OD 600=0.5-0.6), add the IPTG that final concentration is 1mM at once, cultivated 4 hours for 30 ℃, culture is collected bacterium through centrifugal 5 min of 12000 rpm, after thalline washs with the slow liquid of equal-volume 50 mM sodium phosphates, recentrifuge is collected the back and is suspended in pH8.0 with 0.2g fresh weight/ml, contains 50 mM sodium phosphate 300mM NaCl, 10mM imidazoles (imidazole), the 1mM N,O-Diacetylmuramidase is among the proteinase inhibitor DMSF of 2mM.Bacteria suspension (0-4 ℃) ultrasonic disruption at low temperatures is homogenate.Cell homogenates is got supernatant liquor and is got the kfiD crude extract through centrifugal 20 min of 12000 RMP; This crude extract joins in Ni-NTA-Agrose (the Sigma company product) chromatography column, through 50 mM sodium phosphate buffers (pH 8.0), contain 300mM NaCl, the washing of 20 mM imidazoles (imidazole) and the slow liquid (pH 8.0) of 50 mM sodium phosphates, contain 300mM NaCl, 250 mM imidazoles (imidazole) wash-outs, collect elutriant, this solution in 0-4 ℃ of environment, through 10mM pH7.0 Tris-HCl dialysed overnight, purifying
KfID.From Fig. 3 as seen
KfiThe D gene obtains to efficiently express and be purified as a SDS-PAGE band in intestinal bacteria.
KfiThe determination of activity of D adopts detection UDP-GlcA to carry out in external generation, specifically be in cumulative volume 50 μ l systems, final concentration contains 100 mM Tris-HCl buffer (pH 8.8), 1 mM UDP-Glc, 2 mM NAD+, 5 mM dithiothreitol (DTT) (Dithiothreitol), the different purification phase of middle adding 2.5-5 μ l, different dilution
KfiD enzyme liquid, behind 37 ℃ of reaction 10 min, 100 ℃ of 5min termination reactions.Reaction mixture is got supernatant liquor behind centrifugal 10 min of 13000 rpm, and supernatant liquor is used for the generation that HPLC analyzes UDP-GlcA after the cellulose acetate filter of 0.22 μ l filters.
UDP-GlcA analyzes and carries out in high performance liquid chromatography (HPLC) system (Agilent 1100 series), analytical column is carbon C18 reversed-phase column (Waters Jsphere ODS H80,150 x, 4.6 mm i.d., 4 μ m-particle size). the moving phase of analysis is the filtering solution A of cellulose acetate filter (the 100 mM KH through 0.22 μ l
2PO4 pH, 5.3 include 8 mM sulfuric acid tetrabutyl ammonia (tetrabutylammonium hydrogen sulfate) and solution B (70% solution A+30% methyl alcohol (pH 5.9).Chromatography condition is: 40 ℃ of column temperatures, 0 min, 100% solution A, 15min 30%A+70% solution B, 20 min, 100% solution B, 30min 100% solution B, 35min 100% solution A, 50min 100% solution A. use the UDP-GlcA(Sigma product) manufacture typical curve for standard specimen and carry out the calculating that enzymatic UDP-GlcA forms.1 unit of enzyme activity (U) is defined as the enzymatic vigor that 1min catalysis forms 1umol UDP-GlcA.(Bio-Rad, www.biorad.com) company's protein determination kit is a protein standard substance with BSA also to protein content employing Bole in the enzyme liquid, lives with the ratio that calculates enzyme.Table 2 data show that KfiD obtains activity expression in intestinal bacteria.
Remarks: data are the mean+/-standard error of 3 mensuration in the table 2
By table 2 data as can be known
KfiD obtains activity expression in e. coli jm109.
(3)
PmHasWith
KfiThe structure of D gene co-expression carrier in intestinal bacteria:
According to T5:kfiDDNA sequence (SEQ ID NO:9 in the sequence table) the design PCR primer of the pQEkfiD carrier that builds, amplification
T5Promoters driven
KfiThe fragment T5:kfiD that D expresses, concrete primer sequence is:
Upstream primer T5kfiD P1:5'-TA
GAGCTCCCTTTCGTCTTCAC
GSEQ ID NO:7 in the TCGAGA-3'(sequence table), this primer contains
SacThe I site, GAGCTC, wherein subscript G represents C in the original pQE80L carrier is sported G, to eliminate it
XhoThe l site, CTCGAG;
Downstream primer T5kfiD P2:5'-TA
CTCGAGSEQ ID NO:8 in the TTCTGAGGTCATTACTGGATCT-3'(sequence table), this primer contains
XhoI site, CTCGAG;
It is synthetic that primer is given birth to the worker by Shanghai, the masterplate of PCR pQEkfiD carrier, and the parameter of PCR amplification is: 95 ℃, 2min, 1 circulation, 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 1.5min, 25 circulations, 72 ℃, 10 min, 1 circulation.The about 1.4kb amplified production of purifying, at first connect into commercial carrier pGEM-T-Vector and transformed into escherichia coli JM109 bacterial strain, obtain intermediate carrier pT-T5KfiD, SEQ ID NO:9 in the T5:kfiD(sequence table in this carrier) fragment, after sequencing analysis confirmed that nucleotide sequence does not have sudden change, this inserted T5:kfiD fragment warp
SacI and
XhoBehind the I double digestion, connect into plasmid pQEpmHas's
SacI and
SalI site and obtaining
PmHasWith
KfiThe D gene is co-expression carrier pQHK in intestinal bacteria.
(4) structure of expression vector pHK
For making
PmHasWith
KfiD can express in comprising other Gram-negative bacterias of intestinal bacteria, according to dna sequence dna (GenBank No. Y14439) the design primer of the Gram-negative bacteria host wide spectrum plasmid pBBR122 of German MoBiTec company:
Upstream primer PBBR P1:5'-TTTGGT
GTCGACSEQ ID NO:10 in the CTTGCCAGCCCGTGGATATGTGG-3'(sequence table); Contain
SalThe I restriction enzyme site, GTCGAC
Downstream primer PBBR P1:5'-TTAGGT
GTCGACSEQ ID NO:11 in the TCTGTGATGGCTTCCATGTCGGCAG-3'(sequence table), contain
SalThe I restriction enzyme site, GTCGAC);
Increasing with above-mentioned up and down primer comprises its replication site and kalamycin resistance gene among the plasmid pBBR122 in interior fragment, and the parameter of PCR amplification is: 95 ℃, 2 min, 1 circulation; 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 3 min, 25 circulations; 72 ℃, 1 circulation of 10 min, the about 3.2 kb amplified productions of purifying are used
SalBehind the I enzymolysis, add ligase enzyme and connect certainly, self connect product transformed into escherichia coli JM109 bacterial strain, and obtain plasmid pRP (SEQ ID NO:12 in the sequence table) with the kantlex screening by it.In e. coli jm109/pRP, extract plasmid pRP, use
SalConnect into pQE80L behind the I enzymolysis, pQEpmHas's
XhoBehind the I site, obtain plasmid pBQ and pBQpmHas. respectively and will from pT-T5kfiD, use
SacI and
XhoThe T5:kfiD that the I double digestion obtains connects into carrier pBQpmHas's
SacI and
SalThe I site, or will
SalThe pRP fragment of I enzymolysis connects into carrier pQHK's
XhoI site and obtain in Gram-negative bacteria, can express
PmHasWith
KfiD sees Fig. 2 with synthetic hyaluronic expression vector pHK().
Wherein: commercial carrier pGEM-T-Vector is the product of buying from Promega company, the website www.promega.com.cn of the said firm, and Promega company provides the sequence of carrier pGEM-T-Vector.Gram-negative bacteria host wide spectrum plasmid pBBR122 buys from German MoBiTec company, and the dna sequence dna of this Gram-negative bacteria host wide spectrum plasmid pBBR122 is Y14439 in the number of obtaining of the GenBank of NCBI.
(5) efficiently produce hyaluronic engineering colon bacillus pHK/JM109(CGMCC NO:3926) acquisition and evaluation
PHK changes in the e. coli jm109 with this recombinant vectors, promptly obtain the hyaluronic engineering colon bacillus pHK/JM109(CGMCC of described efficient product NO:3926), the evaluation of this bacterial strain and checking, the employing following steps are carried out: rule on the LB flat board that contains 50ug/ml kantlex and 100ug/ml acillin, 37 ℃ grow the single bacterium colony of pHK/JM109, and this list colony inoculation is in the LB of identical antibiotic concentration, 37 ℃, 250rpm shaking table incubated overnight obtains kind of a daughter bacteria.To plant daughter bacteria with 100 times of dilutions (V/V), 37 ℃ when being cultured to OD600 and reaching 0.4-0.5, in nutrient solution, add final concentration and reach the IPTG of 1mM and culture temperature is reduced to 30 ℃, cultivate 6 hours to induce the expression of pmHas and kfiD among the pHK.The bacterial cell of having expressed pmHas and kfiD gene among the pHK is used for through the centrifugal collection of 5000rpm 10 min
PmHasWith
KfiThe Protein Detection of D, enzyme assay and hyaluronic synthetic.Warp
PmHasWith
KfiThe D enzymic activity detects (method as mentioned above) and confirms among the pHK
PmHasWith
KfiThe D gene all can be in intestinal bacteria activity expression (table 1, table 2 and Fig. 3).
Above-described coli strain pHK/JM109 is the efficient hyaluronic acid engineering colon bacillus that produces, and its host strain JM109 is commercially available prod commonly used, can from biological chemistry, molecular biology company (as, Sigma, Promega, worker etc. is given birth in Shanghai) buy and obtain.
Pasteurella multocida (
Pasteurella multocida ubsp. Multocida) strains A TCC 15742 is commercial bacterial strain, is that the preserving number of buying from American type culture collection (being called for short ATCC) is 15742;
The design of primers of amplification is with reference to De Angelis PL etc., 1998 (DeAngelis, P.L et al, Identification and Molecular Cloning of a Unique Hyaluronan Synthase from Pasteurella multocida.J.Biol, Chem. 273,8454 – 8458) Bao Dao sequence designs, and this sequence website sequence number of (NCBI) in the American National bioinformation is AF036004.
Commercial carrier pQE80L is from German Qiagen company (www.qiagen.com, order phone: 800-426-8157) buy, and this www.qiagen.com has announced the sequence of carrier pQE80L.
Sequence table
SEQUENCE LISTING
<110〉Yunnan Province's microbial fermentation engineering research centre company limited
<120〉efficiently produce the hyaluronic acid engineering colon bacillus
<130> /
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 2919
<212> DNA
<213> Pasteurella multocida subsp. Multocida(ATCC15742)
<400> 1
atgaatacat tatcacaagc aataaaagca tataacagca atgactatca attagcactc 60
aaattatttg aaaagtcggc ggaaatctat ggacggaaaa ttgttgaatt tcaaattacc 120
aaatgcaaag aaaaactctc agcacatcct tctgttaatt cagcacatct ttctgtaaat 180
aaagaagaaa aagtcaatgt ttgcgatagt ccgttagata ttgcaacaca actgttactt 240
tccaacgtaa aaaaattagt actttctgac tcggaaaaaa acacgttaaa aaataaatgg 300
aaattgctca ctgagaagaa atctgaaaat gcggaggtaa gagcggtcgc ccttgtacca 360
aaagattttc ccaaagatct ggttttagcg cctttacctg atcatgttaa tgattttaca 420
tggtacaaaa agcgaaagaa aagacttggc ataaaacctg aacatcaaca tgttggtctt 480
tctattatcg ttacaacatt caatcgacca gcaattttat cgattacatt agcctgttta 540
gtaaaccaaa aaacacatta cccgtttgaa gttatcgtga cagatgatgg tagtcaggaa 600
gatctatcac cgatcattcg ccaatatgaa aataaattgg atattcgcta cgtcagacaa 660
aaagataacg gttttcaagc cagtgccgct cggaatatgg gattacgctt agcaaaatat 720
gactttattg gcttactcga ctgtgatatg gcgccaaatc cattatgggt tcattcttat 780
gttgcagagc tattagaaga tgatgattta acaatcattg gtccaagaaa atacatcgat 840
acacaacata ttgacccaaa agacttctta aataacgcga gtttgcttga atcattacca 900
gaagtgaaaa ccaataatag tgttgccgca aaaggggaag gaacagtttc tctggattgg 960
cgcttagaac aattcgaaaa aacagaaaat ctccgcttat ccgattcgcc tttccgtttt 1020
tttgcggcgg gtaatgttgc tttcgctaaa aaatggctaa ataaatccgg tttctttgat 1080
gaggaattta atcactgggg tggagaagat gtggaatttg gatatcgctt attccgttac 1140
ggtagtttct ttaaaactat tgatggcatt atggcctacc atcaagagcc accaggtaaa 1200
gaaaatgaaa ccgatcgtga agcgggaaaa aatattacgc tcgatattat gagagaaaag 1260
gtcccttata tctatagaaa acttttacca atagaagatt cgcatatcaa tagagtacct 1320
ttagtttcaa tttatatccc agcttataac tgtgcaaact atattcaacg ttgcgtagat 1380
agtgcactga atcagactgt tgttgatctc gaggtttgta tttgtaacga tggttcaaca 1440
gataatacct tagaagtgat caataagctt tatggtaata atcctagggt acgcatcatg 1500
tctaaaccaa atggcggaat agcctcagca tcaaatgcag ccgtttcttt tgctaaaggt 1560
tattacattg ggcagttaga ttcagatgat tatcttgagc ctgatgcagt tgaactgtgt 1620
ttaaaagaat ttttaaaaga taaaacgcta gcttgtgttt ataccactaa tagaaacgtc 1680
aatccggatg gtagcttaat cgctaatggt tacaattggc cagaattttc acgagaaaaa 1740
ctcacaacgg ctatgattgc tcaccacttt agaatgttca cgattagagc ttggcattta 1800
actgatggat tcaatgaaaa aattgaaaat gccgtagact atgacatgtt cctcaaactc 1860
agtgaagttg gaaaatttaa acatcttaat aaaatctgct ataaccgtgt attacatggt 1920
gataacacat caattaagaa acttggcatt caaaagaaaa accattttgt tgtagtcaat 1980
cagtcattaa atagacaagg cataacttat tataattatg acgaatttga tgatttagat 2040
gaaagtagaa agtatatttt caataaaacc gctgaatatc aagaagagat tgatatctta 2100
aaagatatta aaatcatcca gaataaagat gccaaaatcg cagtcagtat tttttatccc 2160
aatacattaa acggcttagt gaaaaaacta aacaatatta ttgaatataa taaaaatata 2220
ttcgttattg ttctacatgt tgataagaat catcttacac cagatatcaa aaaagaaata 2280
ctagccttct atcataaaca tcaagtgaat attttactaa ataatgatat ctcatattac 2340
acgagtaata gattaataaa aactgaggcg catttaagta atattaataa attaagtcag 2400
ttaaatctaa attgtgaata catcattttt gataatcatg acagcctatt cgttaaaaat 2460
gacagctatg cttatatgaa aaaatatgat gtcggcatga atttctcagc attaacacat 2520
gattggatcg agaaaatcaa tgcgcatcca ccatttaaaa agctcattaa aacttatttt 2580
aatgacaatg acttaaaaag tatgaatgtg aaaggggcat cacaaggtat gtttatgacg 2640
tatgcgctag cgcatgagct tctgacgatt attaaagaag tcatcacatc ttgccagtca 2700
attgatagtg tgccagaata taacactgag gatatttggt tccaatttgc acttttaatc 2760
ttagaaaaga aaaccggcca tgtatttaat aaaacatcga ccctgactta tatgccttgg 2820
gaacgaaaat tacaatggac aaatgaacaa attgaaagtg caaaaagagg agaaaatata 2880
cctgttaaca agttcattat taatagtata actctataa 2919
<210> 2
<211> 1179
<212> DNA
<213> Escherichia coli K5(ATCC23500)
<400> 2
atgttcggaa cactaaaaat aactgtttca ggcgctggtt acgttgggct ttcaaatgga 60
attctaatgg ctcaaaatca tgaagtggtt gcatttgata cccatcaaaa aaaagttgac 120
ttacttaatg ataaactctc tcctatagag gataaggaaa ttgaaaatta tctttcaact 180
aaaatactta attttcgcgc aactactaac aaatatgaag cctataaaaa tgccaattac 240
gttattattg ctacaccaac gaattatgac ccaggttcaa attactttga tacatcaagc 300
gttgaagctg tcattcgtga cgtaacggaa atcaacccaa acgcaattat ggtggttaaa 360
tctacggtcc cagtaggttt cacaaaaaca attaaagaac atttaggtat taataatatt 420
atcttctctc cagaattttt acgagaagga agagccctat acgataatct ccatccatct 480
cgcattatta tcggtgaatg ttctgaacgg gcagaacgtt tggcagtgtt atttcaggaa 540
ggagcgatta aacaaaatat acccgtttta tttacagatt ctacggaagc ggaagcgatt 600
aagttatttt caaatactta tttggctatg cgagttgcat tttttaatga attggatagt 660
tacgcagaaa gttttggtct gaatacgcgt cagattattg acggtgtttg tttggatccg 720
cgcattggta attactacaa taatccttct tttggttatg gtggctactg tttgccaaaa 780
gataccaagc aattattagc caactatcag tctgttccga ataaacttat atctgcaatt 840
gttgatgcta accgtacacg taaggacttt atcactaatg ttattttgaa acatagacca 900
caagttgtgg gggtttatcg tttgattatg aaaagtggtt cagataattt tagagattct 960
tctattcttg gtattataaa gcgtatcaag aaaaaaggcg tgaaagtaat tatttatgag 1020
ccgcttattt ctggagatac attctttaac tcacctttgg aacgggagct ggcgatcttt 1080
aaagggaaag ctgatattat tatcactaac cgaatgtcag aggagttgaa cgatgtggtc 1140
gacaaagtct atagtcgcga tttgtttaaa tgtgactaa 1179
<210> 3
<211> 31
<212> DNA
<213〉artificial-synthetic DNA
<400> 3
taggatccat gaacacatta tcacaagcaa t 31
<210> 4
<211> 32
<212> DNA
<213〉artificial-synthetic DNA
<400> 4
tagagctctt atagagttat actattaata at 32
<210> 5
<211> 34
<212> DNA
<213〉artificial-synthetic DNA
<400> 5
tggagatcta tgttcggaac actaaaaata actg 34
<210> 6
<211> 34
<212> DNA
<213〉artificial-synthetic DNA
<400> 6
ttctcgagtt agtcacattt aaacaaatcg cgac 34
<210> 7
<211> 29
<212> DNA
<213〉artificial-synthetic DNA
<400> 7
tagagctccc tttcgtcttc acgtcgaga 29
<210> 8
<211> 30
<212> DNA
<213〉artificial-synthetic DNA
<400> 8
tactcgagtt ctgaggtcat tactggatct 30
<210> 9
<211> 1423
<212> DNA
<213〉engineering makes up fragment
<400> 9
tagagctccc tttcgtcttc acgtcgagaa atcataaaaa atttatttgc tttgtgagcg 60
gataacaatt ataatagatt caattgtgag cggataacaa tttcacacag aattcattaa 120
agaggagaaa ttaactatga gaggatcgca tcaccatcac catcacggat ctatgttcgg 180
aacactaaaa ataactgttt caggcgctgg ttacgttggg ctttcaaatg gaattctaat 240
ggctcaaaat catgaagtgg ttgcatttga tacccatcaa aaaaaagttg acttacttaa 300
tgataaactc tctcctatag aggataagga aattgaaaat tatctttcaa ctaaaatact 360
taattttcgc gcaactacta acaaatatga agcctataaa aatgccaatt acgttattat 420
tgctacacca acgaattatg acccaggttc aaattacttt gatacatcaa gcgttgaagc 480
tgtcattcgt gacgtaacgg aaatcaaccc aaacgcaatt atggtggtta aatctacggt 540
cccagtaggt ttcacaaaaa caattaaaga acatttaggt attaataata ttatcttctc 600
tccagaattt ttacgagaag gaagagccct atacgataat ctccatccat ctcgcattat 660
tatcggtgaa tgttctgaac gggcagaacg tttggcagtg ttatttcagg aaggagcgat 720
taaacaaaat atacccgttt tatttacaga ttctacggaa gcggaagcga ttaagttatt 780
ttcaaatact tatttggcta tgcgagttgc attttttaat gaattggata gttacgcaga 840
aagttttggt ctgaatacgc gtcagattat tgacggtgtt tgtttggatc cgcgcattgg 900
taattactac aataatcctt cttttggtta tggtggctac tgtttgccaa aagataccaa 960
gcaattatta gccaactatc agtctgttcc gaataaactt atatctgcaa ttgttgatgc 1020
taaccgtaca cgtaaggact ttatcactaa tgttattttg aaacatagac cacaagttgt 1080
gggggtttat cgtttgatta tgaaaagtgg ttcagataat tttagagatt cttctattct 1140
tggtattata aagcgtatca agaaaaaagg cgtgaaagta attatttatg agccgcttat 1200
ttctggagat acattcttta actcaccttt ggaacgggag ctggcgatct ttaaagggaa 1260
agctgatatt attatcacta accgaatgtc agaggagttg aacgatgtgg tcgacaaagt 1320
ctatagtcgc gatttgttta aatgtgacta actcgaccca agcttaatta gctgagcttg 1380
gactcctgtt gatagatcca gtaatgacct cagaactcga gta 1423
<210> 10
<211> 33
<212> DNA
<213〉artificial-synthetic DNA
<400> 10
tggtgtcgac cttgccagcc cgtggatatg tgg 33
<210> 11
<211> 35
<212> DNA
<213〉artificial-synthetic DNA
<400> 11
aggtgtcgac tctgtgatgg cttccatgtc ggcag 35
<210> 12
<211> 3266
<212> DNA
<213> pBBR122
<400> 12
gtcgaccttg ccagcccgtg gatatgtgga cgatggccgc gagcggccac cggctggctc 60
gcttcgctcg gcccgtggac aaccctgctg gacaagctga tggacaggct gcgcctgccc 120
acgagcttga ccacagggat tgcccaccgg ctacccagcc ttcgaccaca tacccaccgg 180
ctccaactgc gcggcctgcg gccttgcccc atcaattttt ttaattttct ctggggaaaa 240
gcctccggcc tgcggcctgc gcgcttcgct tgccggttgg acaccaagtg gaaggcgggt 300
caaggctcgc gcagcgaccg cgcagcggct tggccttgac gcgcctggaa cgacccaagc 360
ctatgcgagt gggggcagtc gaaggcgaag cccgcccgcc tgccccccga gacctgcagg 420
gggggggggg cgctgaggtc tgcctcgtga agaaggtgtt gctgactcat accaggcctg 480
aatcgcccca tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta 540
ggtggaccag ttggtgattt tgaacttttg ctttgccacg gaacggtctg cgttgtcggg 600
aagatgcgtg atctgatcct tcaactcagc aaaagttcga tttattcaac aaagccgccg 660
tcccgtcaag tcagcgtaat gctctgccag tgttacaacc aattaaccaa ttctgattag 720
aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca 780
tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg 840
atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt 900
aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa 960
tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca 1020
ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc 1080
tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc 1140
aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct 1200
tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca 1260
ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt 1320
ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac 1380
tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta 1440
tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc 1500
gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa 1560
gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga 1620
ttttgagaca caacgtggct ttcccccccc cccctgcagg tcccgagcct cacggcggcg 1680
agtgcggggg ttccaagggg gcagcgccac cttgggcaag gccgaaggcc gcgcagtcga 1740
tcaacaagcc ccggaggggc cactttttgc cggaggggga gccgcgccga aggcgtgggg 1800
gaaccccgca ggggtgccct tctttgggca ccaaagaact agatataggg cgaaatgcga 1860
aagacttaaa aatcaacaac ttaaaaaagg ggggtacgca acagctcatt gcggcacccc 1920
ccgcaatagc tcattgcgta ggttaaagaa aatctgtaat tgactgccac ttttacgcaa 1980
cgcataattg ttgtcgcgct gccgaaaagt tgcagctgat tgcgcatggt gccgcaaccg 2040
tgcggcaccc taccgcatgg agataagcat ggccacgcag tccagagaaa tcggcattca 2100
agccaagaac aagcccggtc actgggtgca aacggaacgc aaagcgcatg aggcgtgggc 2160
cgggcttatt gcgaggaaac ccacggcggc aatgctgctg catcacctcg tggcgcagat 2220
gggccaccag aacgccgtgg tggtcagcca gaagacactt tccaagctca tcggacgttc 2280
tttgcggacg gtccaatacg cagtcaagga cttggtggcc gagcgctgga tctccgtcgt 2340
gaagctcaac ggccccggca ccgtgtcggc ctacgtggtc aatgaccgcg tggcgtgggg 2400
ccagccccgc gaccagttgc gcctgtcggt gttcagtgcc gccgtggtgg ttgatcacga 2460
cgaccaggac gaatcgctgt tggggcatgg cgacctgcgc cgcatcccga ccctgtatcc 2520
gggcgagcag caactaccga ccggccccgg cgaggagccg cccagccagc ccggcattcc 2580
gggcatggaa ccagacctgc cagccttgac cgaaacggag gaatgggaac ggcgcgggca 2640
gcagcgcctg ccgatgcccg atgagccgtg ttttctggac gatggcgagc cgttggagcc 2700
gccgacacgg gtcacgctgc cgcgccggta gcacttgggt tgcgcagcaa cccgtaagtg 2760
cgctgttcca gactatcggc tgtagccgcc tcgccgccct ataccttgtc tgcctccccg 2820
cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg aatggaagcc ggcggcacct 2880
cgctaacgga ttcaccgttt ttatcaggct ctgggaggca gaataaatga tcatatcgtc 2940
aattattacc tccacgggga gagcctgagc aaactggcct caggcatttg agaagcacac 3000
ggtcacactg cttccggtag tcaataaacc ggtaaaccag caatagacat aagcggctat 3060
ttaacgaccc tgccctgaac cgacgaccgg gtcgaatttg ctttcgaatt tctgccattc 3120
atccgcttat tatacttatt caggcgtagc accaggcgtt taagggcacc aataactgcc 3180
ttaaaaaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt cattaagcat 3240
tctgccgaca tggaagccat cacaga 3266
Claims (2)
1. efficient hyaluronic acid engineering colon bacillus that produces, it is characterized in that its classification called after colon bacillus (
Escherichia coli), preserving number is CGMCC No.3926.
2. the preparation method of the described efficient product hyaluronic acid engineering colon bacillus of claim 1 is characterized in that carrying out according to the following steps:
(1) make up the expression vector pQEpmHas contain hyaluronan synthase gene, with the pcr amplification method clone be derived from Pasteurella multocida (
Pasteurella multocidaSubsp.
Multocida) in hyaluronan synthase gene (
PAsteurella
mUltocida
hYaluronic
aCid synthase
PmHas), the design of its pcr amplification clone the primer is as follows:
Upstream primer pmHas P1 is 5'-TA
GGATCC ATGSEQ ID No:3 in the AACACATTATCACAAGCAAT-3'(sequence table), contain
BamH I site GGATCC and initiator codon ATG;
Downstream primer is
PmHasP2 is 5'-TA
GAGCTC TTASEQ ID No:4 in the TAGAGTTATACTATTAATAAT-3'(sequence table), contain
SacI site GAGCTC and terminator codon TAA;
The masterplate of PCR is the genomic dna of Pasteurella multocida strains A TCC 15742, the amplified production warp
BamH I and
SacBehind the I double digestion, connect into the identical restriction enzyme site among the commercial carrier pQE80L, insert fragment after DNA order-checking conclusive evidence does not have sudden change, with obtaining carrier called after pQEpmHas, gene is
PmHas(SEQ ID No:1 in the sequence table);
(2) make up the expression vector pQEkfiD contain the uridine diphosphoglucose dehydrogenase gene, with the pcr amplification method clone be derived from come from intestinal bacteria (
Escherichia coli, E.coli) GDPG dehydrogenase gene (K among the bacterial strain k5
5Capsule gene D,
KfiD), the design of primers of amplification is as follows:
Upstream primer
KfiD P1:5'-TGG
AGATCT ATGSEQ ID No:5 in the TTCGGAACACTAAAAATAACT G-3'(sequence table), contain Bgl II site AGATCT and initiator codon ATG);
Downstream primer
KfiD P2:5'-TT
CTCGAG TTASEQ ID No:6 in the GTCACATTTAAA CAAATCGCGAC-3'(sequence table), contain
XhoI site CTCGAG and terminator codon TAA;
The masterplate of PCR is the genomic dna of intestinal bacteria bacterium k5 strains A TCC 23500, the also product of purifying that successfully increases, warp
BglII and
XhoBehind the I double digestion, connect into the commercial carrier pQE80L's of Qiagen company
BamH I and
SalI site, gained carrier after DNA sequencing analysis conclusive evidence is inserted fragment and is not had sudden change, the carrier called after pQEkfiD that obtains, corresponding gene called after
KfiD gene (SEQ ID No:2 in the sequence table);
(3) make up co expression hyaluronan synthase gene and the recombinant vectors that contains the uridine diphosphoglucose dehydrogenase gene in the intestinal bacteria:
According to the DNA sequences Design PCR primer of the pQEkfiD carrier that builds, amplification T5 promoters driven
KfiThe fragment T5:kfiD that D expresses, the primer sequence design is as follows:
Upstream primer T5kfiD P1:5'-TA
GAGCTCCCTTTCGTCTTCAC
GSEQ ID No:7 in the TCGAGA-3'(sequence table), this primer contains
SacThe I site, GAGCTC, wherein subscript G represents C in the original pQE80L carrier is sported G, to eliminate it
XhoThe l site, CTCGAG;
Downstream primer T5kfiD P2:5'-TA
CTCGAGSEQ ID No:8 in the TTCTGAGGTCATTACTGGATCT-3'(sequence table), this primer contains
XhoI site, CTCGAG;
The masterplate of PCR pQEkfiD carrier, the T5:kfiD amplified production of purifying, at first connect into pGEM-T-Vector and transformed into escherichia coli JM109 bacterial strain, obtain intermediate carrier pT-T5KfiD, SEQ ID No:9 in the T5:kfiD(sequence table in this carrier) fragment, after sequencing analysis confirmed that nucleotide sequence does not have sudden change, this inserted pT-T5KfiD fragment warp
SacI and
XhoBehind the I double digestion, connect into expression vector pQEpmHas's
SacI and
SalI site and obtaining
PmHasWith
KfiThe recombinant vectors pQHK of D gene coexpression in intestinal bacteria;
(4) structure of expression vector pHK
Dna sequence dna (GenBank No. Y14439) design primer with the Gram-negative bacteria host wide spectrum plasmid pBBR122 of German MoBiTec company:
Upstream primer PBBR P1:5'-TTTGGT
GTCGACSEQ ID NO:10 in the CTTGCCAGCCCGTGGATATGTGG-3'(sequence table); Contain
SalThe I restriction enzyme site, GTCGAC;
Downstream primer PBBR P1:5'-TTAGGT
GTCGACSEQ ID NO:11 in the TCTGTGATGGCTTCCATGTCGGCAG-3'(sequence table), contain
SalThe I restriction enzyme site, GTCGAC;
With comprising its replication site and kalamycin resistance gene among the above-mentioned primer amplification plasmid pBBR122 in interior fragment, the parameter of PCR amplification is: 95 ℃, 2 min, 1 circulation; 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 3 min, 25 circulations; 72 ℃, 1 circulation of 10 min, purifying 3.2 kb amplified productions are used
SalBehind the I enzymolysis, add ligase enzyme and connect certainly, self connect product transformed into escherichia coli JM109 bacterial strain by it, and obtain plasmid pRP (SEQ ID NO:12 in the sequence table) with kantlex screening, extract plasmid pRP, behind the SalI enzymolysis, connect into above-mentioned can be in intestinal bacteria coexpression pmHas and
KfiThe pQHK's of D gene
XhoThe I site obtains coexpression in Gram-negative bacteria
PmHasWith
KfiD synthesizes hyaluronic expression vector pHK;
(5) this recombinant vectors pHK is changed over to the hyaluronic engineering colon bacillus pHK/JM109 of the described efficient product of acquisition CGMCC NO:3926 in the e. coli jm109.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201110006213A CN102154190B (en) | 2011-01-13 | 2011-01-13 | Engineering escherichia coli capable of efficiently producing hyaluronic acid and preparation method thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201110006213A CN102154190B (en) | 2011-01-13 | 2011-01-13 | Engineering escherichia coli capable of efficiently producing hyaluronic acid and preparation method thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN102154190A true CN102154190A (en) | 2011-08-17 |
CN102154190B CN102154190B (en) | 2012-09-05 |
Family
ID=44435865
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201110006213A Active CN102154190B (en) | 2011-01-13 | 2011-01-13 | Engineering escherichia coli capable of efficiently producing hyaluronic acid and preparation method thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN102154190B (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103352044A (en) * | 2013-07-05 | 2013-10-16 | 安徽农业大学 | Obtaining method for building Spot-9-UDP-glucose dehydrogenase gene segments |
CN106381279A (en) * | 2016-08-29 | 2017-02-08 | 中国药科大学 | Extracellular polymeric substances, preparation method and application thereof |
CN110093367A (en) * | 2019-05-17 | 2019-08-06 | 汪洋 | A kind of bacillus subtilis oscillation mode gene expression system and its construction method and application |
CN110923173A (en) * | 2019-12-26 | 2020-03-27 | 江南大学 | Enterobacter and application thereof |
CN111040980A (en) * | 2016-07-18 | 2020-04-21 | 清华大学 | Recombinant corynebacterium glutamicum for high-yield low-molecular-weight hyaluronic acid and construction method and application thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101633931A (en) * | 2008-07-25 | 2010-01-27 | 山东省生物药物研究院 | Hyaluronidase expression vector and application thereof |
-
2011
- 2011-01-13 CN CN201110006213A patent/CN102154190B/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101633931A (en) * | 2008-07-25 | 2010-01-27 | 山东省生物药物研究院 | Hyaluronidase expression vector and application thereof |
Non-Patent Citations (4)
Title |
---|
《Biotechnology and Bioengineering》 20081101 Huimin Yu等 A High-Throughput Screen for Hyaluronic Acid Accumulation in Recombinant Escherichia coli Transformed by Libraries of Engineered Sigma Factors 全文 1-2 第101卷, 第4期 * |
《Metabolic Engineering》 20081231 Huimin Yu等 Metabolic engineering of Escherichia coli for biosynthesis of hyaluronic acid 全文 1-2 第10卷, * |
《中国生物工程杂志》 20081231 易梅等 透明质酸的应用及制备方法 全文 1-2 第28卷, 第6s期 * |
《山东生物化学与分子生物学会2009年学术会议论文汇编》 20091231 刘飞等 兽疫链球菌透明质酸酶在大肠杆菌中的高效表达和酶学性质 全文 1-2 , * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103352044A (en) * | 2013-07-05 | 2013-10-16 | 安徽农业大学 | Obtaining method for building Spot-9-UDP-glucose dehydrogenase gene segments |
CN111040980A (en) * | 2016-07-18 | 2020-04-21 | 清华大学 | Recombinant corynebacterium glutamicum for high-yield low-molecular-weight hyaluronic acid and construction method and application thereof |
CN111040980B (en) * | 2016-07-18 | 2021-09-03 | 清华大学 | Recombinant corynebacterium glutamicum for high-yield low-molecular-weight hyaluronic acid and construction method and application thereof |
CN106381279A (en) * | 2016-08-29 | 2017-02-08 | 中国药科大学 | Extracellular polymeric substances, preparation method and application thereof |
CN106381279B (en) * | 2016-08-29 | 2019-11-05 | 中国药科大学 | A kind of extracellular polysaccharide of bacteria, preparation method and its application |
CN110093367A (en) * | 2019-05-17 | 2019-08-06 | 汪洋 | A kind of bacillus subtilis oscillation mode gene expression system and its construction method and application |
CN110093367B (en) * | 2019-05-17 | 2021-11-19 | 汪洋 | Bacillus subtilis oscillatory type gene expression system and construction method and application thereof |
CN110923173A (en) * | 2019-12-26 | 2020-03-27 | 江南大学 | Enterobacter and application thereof |
CN110923173B (en) * | 2019-12-26 | 2021-10-08 | 江南大学 | Enterobacter and application thereof |
Also Published As
Publication number | Publication date |
---|---|
CN102154190B (en) | 2012-09-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108929878B (en) | Coding gene of alginate lyase and application thereof | |
McIntosh et al. | Curdlan and other bacterial (1→ 3)-β-D-glucans | |
CN101608166B (en) | Flavobacterium strain and application thereof in generating agarase | |
CN102154190B (en) | Engineering escherichia coli capable of efficiently producing hyaluronic acid and preparation method thereof | |
CN105431534A (en) | beta-1,3-glucanase, polynucleotide, recombinant vector, transformant, production method for beta-1,3-glucanase, enzyme preparation, and production method for paramylon having reduced molecular weight | |
CN113684157B (en) | Bacillus subtilis for producing levansucrase and application thereof | |
US5482843A (en) | Enzyme of use in chitosan hydrolysis | |
US20130337541A1 (en) | Thermostable chitosanase | |
CN102154360B (en) | Recombinant expression vectors pQHK and pHK producing hyaluronic acid and construction method thereof | |
CN102154405B (en) | Preparation method of hyaluronic acid | |
KR20120078421A (en) | Strain of flammeovirga sp. having a agar-decomposition activity and method of producing agarooligosaccharides using the same | |
CN111334488B (en) | Laminarin enzyme OUC-L1, and coding gene and application thereof | |
CN102732456B (en) | Organic solvent-resistant glycosidase Fru6, and mutant and application thereof | |
JP2023534879A (en) | N-Acetylglucosamine-producing strain and method for construction and use thereof | |
CN116064484B (en) | Mutant chitinase SsChi18A-2 and application thereof | |
CN116640744A (en) | Chitosanase OUC-CsnA4-S49I, application thereof and method for preparing chitosan oligosaccharide | |
CN114457057B (en) | Chitosan mutant and application thereof | |
Ahmed et al. | Hyaluronic acid production by Klebsiella pneumoniae strain H15 (OP354286) under different fermentation conditions | |
CN112458022B (en) | Bacillus licheniformis Bl22 for high yield of chitin deacetylase and related products and application thereof | |
CN104087604A (en) | Genetic expression sequence of inulin fructotransferase | |
Al-Rmedh et al. | Curdlan Gum, Properties, Benefits and Applications | |
JPWO2002086116A1 (en) | Sulfated fucoglucuronomannan | |
CN116064485B (en) | Mutant chitinase SsChi18A-3 and application thereof | |
CN113493811B (en) | Method for preparing laminariae disaccharide by using cellulose as substrate | |
Wannasutta et al. | Identification of endophytic actinobacteria from Jerusalem artichoke and examination of inulinase gene and enzyme properties |
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 | ||
PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: An efficient hyaluronic acid producing engineering Escherichia coli and its preparation method Effective date of registration: 20231120 Granted publication date: 20120905 Pledgee: Kunming Dongfeng Sub branch of Bank of China Ltd. Pledgor: MICROBIAL FERMENTATION ENGINEERING RESEARCH CENTER Co.,Ltd. OF YUNNAN PROVINCE Registration number: Y2023530000066 |