CN101831452B - Method for efficiently expressing and producing T4 lysozyme through recombinant trichoderma reesei in inductive mode - Google Patents
Method for efficiently expressing and producing T4 lysozyme through recombinant trichoderma reesei in inductive mode Download PDFInfo
- Publication number
- CN101831452B CN101831452B CN2010101584539A CN201010158453A CN101831452B CN 101831452 B CN101831452 B CN 101831452B CN 2010101584539 A CN2010101584539 A CN 2010101584539A CN 201010158453 A CN201010158453 A CN 201010158453A CN 101831452 B CN101831452 B CN 101831452B
- Authority
- CN
- China
- Prior art keywords
- mould
- gene
- rui shi
- sequence
- wood
- 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.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims abstract description 48
- 101000925646 Enterobacteria phage T4 Endolysin Proteins 0.000 title claims abstract description 38
- 230000001939 inductive effect Effects 0.000 title claims abstract description 17
- 241000499912 Trichoderma reesei Species 0.000 title abstract description 13
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 78
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 42
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 42
- 108010008885 Cellulose 1,4-beta-Cellobiosidase Proteins 0.000 claims abstract description 38
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 31
- 241000894006 Bacteria Species 0.000 claims abstract description 20
- 108020004705 Codon Proteins 0.000 claims abstract description 20
- 239000013612 plasmid Substances 0.000 claims abstract description 14
- 238000000855 fermentation Methods 0.000 claims abstract description 13
- 230000004151 fermentation Effects 0.000 claims abstract description 13
- 239000002023 wood Substances 0.000 claims description 98
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 claims description 68
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 28
- 101150084750 1 gene Proteins 0.000 claims description 26
- 239000002773 nucleotide Substances 0.000 claims description 26
- 125000003729 nucleotide group Chemical group 0.000 claims description 26
- 235000018102 proteins Nutrition 0.000 claims description 23
- 239000007788 liquid Substances 0.000 claims description 22
- 108020004414 DNA Proteins 0.000 claims description 20
- 230000008569 process Effects 0.000 claims description 19
- 230000008521 reorganization Effects 0.000 claims description 19
- 241000223259 Trichoderma Species 0.000 claims description 17
- 239000013604 expression vector Substances 0.000 claims description 15
- 230000003248 secreting effect Effects 0.000 claims description 15
- 230000008859 change Effects 0.000 claims description 14
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 13
- 238000012408 PCR amplification Methods 0.000 claims description 11
- 108091026890 Coding region Proteins 0.000 claims description 10
- 108700007698 Genetic Terminator Regions Proteins 0.000 claims description 10
- 239000013613 expression plasmid Substances 0.000 claims description 9
- 230000006698 induction Effects 0.000 claims description 9
- 238000011081 inoculation Methods 0.000 claims description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 8
- 229920002678 cellulose Polymers 0.000 claims description 8
- 239000013600 plasmid vector Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 108091008146 restriction endonucleases Proteins 0.000 claims description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 7
- 239000001913 cellulose Substances 0.000 claims description 7
- 239000012634 fragment Substances 0.000 claims description 7
- 229910052760 oxygen Inorganic materials 0.000 claims description 7
- 239000001301 oxygen Substances 0.000 claims description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 5
- 206010001497 Agitation Diseases 0.000 claims description 4
- 238000013019 agitation Methods 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 102000003960 Ligases Human genes 0.000 claims description 3
- 108090000364 Ligases Proteins 0.000 claims description 3
- 238000006062 fragmentation reaction Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- 239000013598 vector Substances 0.000 claims description 3
- 108091029865 Exogenous DNA Proteins 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 claims description 2
- 238000013461 design Methods 0.000 claims description 2
- 230000029087 digestion Effects 0.000 claims description 2
- 238000013467 fragmentation Methods 0.000 claims description 2
- 238000012423 maintenance Methods 0.000 claims description 2
- 230000035800 maturation Effects 0.000 claims description 2
- 238000012163 sequencing technique Methods 0.000 claims description 2
- 238000011160 research Methods 0.000 abstract description 6
- 230000012010 growth Effects 0.000 abstract description 5
- 230000004071 biological effect Effects 0.000 abstract description 4
- 235000013305 food Nutrition 0.000 abstract description 4
- 239000000284 extract Substances 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 239000000047 product Substances 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 11
- 108090000790 Enzymes Proteins 0.000 description 11
- 229940088598 enzyme Drugs 0.000 description 11
- 230000000694 effects Effects 0.000 description 9
- 239000012530 fluid Substances 0.000 description 9
- 239000011780 sodium chloride Substances 0.000 description 9
- 241000233866 Fungi Species 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 8
- 239000002609 medium Substances 0.000 description 7
- 235000010980 cellulose Nutrition 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 6
- 230000006801 homologous recombination Effects 0.000 description 6
- 238000002744 homologous recombination Methods 0.000 description 6
- 239000008176 lyophilized powder Substances 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 230000009466 transformation Effects 0.000 description 6
- 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 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 241000192041 Micrococcus Species 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 101150052795 cbh-1 gene Proteins 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000013016 damping Methods 0.000 description 5
- 230000008021 deposition Effects 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- MEIRRNXMZYDVDW-MQQKCMAXSA-N (2E,4E)-2,4-hexadien-1-ol Chemical compound C\C=C\C=C\CO MEIRRNXMZYDVDW-MQQKCMAXSA-N 0.000 description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 4
- 235000019257 ammonium acetate Nutrition 0.000 description 4
- 230000004087 circulation Effects 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 3
- 239000005695 Ammonium acetate Substances 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 229940043376 ammonium acetate Drugs 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 230000000855 fungicidal effect Effects 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000007500 overflow downdraw method Methods 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000001742 protein purification Methods 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 102100036826 Aldehyde oxidase Human genes 0.000 description 2
- 108010059892 Cellulase Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 241000196324 Embryophyta Species 0.000 description 2
- 101000928314 Homo sapiens Aldehyde oxidase Proteins 0.000 description 2
- 108090000604 Hydrolases Proteins 0.000 description 2
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 2
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 2
- 241000223261 Trichoderma viride Species 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 229940106157 cellulase Drugs 0.000 description 2
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000002070 germicidal effect Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000002331 protein detection Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 241000838140 Acrophialophora fusispora Species 0.000 description 1
- 244000189799 Asimina triloba Species 0.000 description 1
- 235000006264 Asimina triloba Nutrition 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 229920002498 Beta-glucan Polymers 0.000 description 1
- 240000002791 Brassica napus Species 0.000 description 1
- 235000011293 Brassica napus Nutrition 0.000 description 1
- 235000000540 Brassica rapa subsp rapa Nutrition 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 235000009467 Carica papaya Nutrition 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 206010010254 Concussion Diseases 0.000 description 1
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 101000955144 Drosophila melanogaster WASH complex subunit 1 Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 102000008791 Lysozyme C Human genes 0.000 description 1
- 102100033468 Lysozyme C Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000183011 Melanocarpus Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000191938 Micrococcus luteus Species 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase 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
- XUYPXLNMDZIRQH-LURJTMIESA-N N-acetyl-L-methionine Chemical compound CSCC[C@@H](C(O)=O)NC(C)=O XUYPXLNMDZIRQH-LURJTMIESA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-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
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 244000088415 Raphanus sativus Species 0.000 description 1
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000005267 amalgamation Methods 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002086 displacement chromatography Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 108010079557 endopeptidase B Proteins 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- YQYJSBFKSSDGFO-FWAVGLHBSA-N hygromycin A Chemical compound O[C@H]1[C@H](O)[C@H](C(=O)C)O[C@@H]1Oc1ccc(\C=C(/C)C(=O)N[C@@H]2[C@@H]([C@H]3OCO[C@H]3[C@@H](O)[C@@H]2O)O)cc1O YQYJSBFKSSDGFO-FWAVGLHBSA-N 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009655 industrial fermentation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000001696 ion exchange chromatographie Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 238000001728 nano-filtration Methods 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 101710135378 pH 6 antigen Proteins 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000004763 spore germination Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 210000001138 tear Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 108010046845 tryptones Proteins 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method for efficiently expressing and producing recombinant protein of T4 lysozyme in trichoderma reesei cell in an inductive mode. The method comprises the following steps: 1) increasing the biological output of T4 lysozyme genes in trichoderma reesei engineering bacteria by using the synthetic T4 lysozyme gene with optimized codon; 2) taking the sequence of a cellobiohydrolase gene promoter or terminator derived from the trichoderma reesei as a homologous sequence for completely integrating exogenous plasmid into the trichoderma reesei genome; 3) adjusting and controlling the high-efficiency expression of the exogenous gene of the T4 lysozyme gene in the trichoderma reesei in the inductive mode by using trichoderma reesei cellobiohydrolase gene promoter; and 4) providing a specific trichoderma reesei engineering bacteria fermentation culture and growth condition so as to improve the biological output of recombinant protein of T4 lysozyme and quickly extract the recombinant exogenous protein. The pure recombinant protein of T4 lysozyme prepared by the method has biological activity and can be widely applied in fields such as medicinal treatment, foods, feeds, scientific research and the like.
Description
Technical field
The present invention relates to the genetically engineered field, particularly relate to a kind of method of in Rui Shi wood is mould, expressing and producing T4 N,O-Diacetylmuramidase recombinant protein with the induction type mode.
Background technology
N,O-Diacetylmuramidase (Lysozyme; EC3.2.17) be meant that one type extensively is present in occurring in nature and multiple Gram-positive and negative pathogenetic bacteria, fungi and some virus had and kills or inhibiting hydrolase, nineteen twenty-two is found by Britain bacteriologist Fleming first.The main sterilization mode of this lytic enzyme is-acetylmuramic acid and the β-1 between the N-acetylglucosamine that destroys in the bacteria cell wall; 4 glycosidic links cause cell wall to break and Dissolve things inside oozes out, and cause bacterium collapse and dead thus; Therefore, the N,O-Diacetylmuramidase-acetylmuramic acid enzyme that is otherwise known as.At occurring in nature, the source of N,O-Diacetylmuramidase is very widely, is broadly divided into several types of animal, plant and microbe-derived N,O-Diacetylmuramidases etc.The N,O-Diacetylmuramidase of animal-origin generally is present in people and mammiferous multiple tissue and the secretory product, as it is medium to be present in tears, saliva, liver, kidney, Lymphoid tissue and egg white.Also can be separated to N,O-Diacetylmuramidase in the plants such as pawpaw, turnip and radish, they are considered in the first road defensive barrier of biological human body, play an important role.
In multiple microbe body, found the existence of N,O-Diacetylmuramidase at present, like styreptomyces globispotus strain, acetone clostridium butylicum and multiple phage etc.The T4 N,O-Diacetylmuramidase derives from and infects colibacillary T4 phage, by T4 phage gpe coded by said gene (Arisaka F, et al., 2003), and 495 bases of full length gene, 164 amino acid of encoding, molecular weight 18700Da.When phage particle after the host cell internal packing is accomplished, under the effect of T4 N,O-Diacetylmuramidase, cause host-Bacillus coli cells cracking.Up to now, the T4 N,O-Diacetylmuramidase has had 30 years of researches historical, and the crystalline structure of this enzyme and various mutations body thereof has obtained resolving.The T4 N,O-Diacetylmuramidase is made up of two structural domains: the α/beta structure territory and the αLuo Xuanjiegou territory that is positioned at the C end that are positioned at the N end.Discover; The germicidal action of T4 N,O-Diacetylmuramidase mainly shows two aspects: the one, and this enzyme has the-acetylmuramic acid enzymic activity; The 2nd, being positioned at the positively charged α helical domain of this enzyme C end can combine with bacterium or the electronegative cytolemma of fungi, thereby disturbs the normal physiological metabolism of host cell.The αLuo Xuanjiegou territory that is positioned at T4 lysozyme C end mainly contains 4, is respectively α 1 (115-122), α 2 (126-134), α 3 (137-141) and α 4 (143-155).The α 2-α 3 and α 4 small peptides of synthetic do not have the-acetylmuramic acid enzymic activity, but still the biological activity of performance antibacterium or fungi.With after the T4 N,O-Diacetylmuramidase heat denatured, lose equally, but still keep stronger sterilizing ability though find its-acetylmuramic acid enzyme bioactivity.Research shows, the T4 N,O-Diacetylmuramidase is carried out structure of modification can improve its fungicidal activity and thermostability with modifying, and for example to have the fungicidal activity of the T4 N,O-Diacetylmuramidase of 6 histidine-tagged structures (6xHis-Tag) be 2 times of natural T4 N,O-Diacetylmuramidase to albumen n end; In addition; After holding the 6th amino acids to become the Methionin K of positively charged N by hydrophobic methionine(Met) M; Anti-microbial activity can improve 4 times; But the stability decreases of protein molecular is inferred that the reason that its fungicidal activity improves possibly be because C end degree of freedom strengthens, thereby the αLuo Xuanjiegou territory can more effectively be combined with the cytolemma target.
The T4 N,O-Diacetylmuramidase is except can killing the several diseases indigenous bacteria, and is also inhibited to some pathogenic fungi.Though the T4 N,O-Diacetylmuramidase can not pass the cytolemma of fungus conidium, the fungus conidium after this enzyme is handled no longer expands and germinates.The T4 N,O-Diacetylmuramidase also has certain destruction to potato protoplastis adventitia, but research confirms that to people and mammiferous cell toxicological harmless effect therefore, the T4 N,O-Diacetylmuramidase all has great application potential at industrial circles such as medicine, food and feeds.
From the Bacillus coli cells of T4 phage-infect, extract the T4 N,O-Diacetylmuramidase, production technique is not only loaded down with trivial details, yield poorly, and is difficult to industriallization.In recent years; Fast development along with molecular biology and recombinant gene; The method that makes up expression such as engineering strain such as yeast and intestinal bacteria and production external source recombinant protein more and more receives scientific worker and industrial circle expert's favor; Filamentous fungus then has many advantages equally as the new host of exogenous protein expression, as: 1) foreign protein genes also can be incorporated in the filamentous fungus genome and can obtain the transformant of genetic stability; 2) strong promoter that closely regulated and control by inductor can start efficiently expressing of downstream foreign protein genes, and can recombinant exogenous protein be secreted into outside the born of the same parents, thereby helps the purifying work in later stage; 3) can translate post-treatment and modification to the external source recombinant protein as eukaryotic expression system, comprise generation and glycosylation of disulfide linkage etc.; 4) thalline and its meta-bolites have no side effect to human body and Mammals etc.; 5) industrial fermentation technology is ripe, makes things convenient for the suitability for industrialized production of recombinant exogenous protein etc.
Employed Rui Shi wood mould (Trichoderma reesei) is a kind of harmless saprophytic sex pilus shape fungi among the present invention, often is applied to the production of cellobiohydrolase in the industry, and its output reaches as high as 40g/L (Jiang Tian is first-class, 2007).In the cellulase system 1; 4-beta fibers disaccharide-hydrolysing enzymes 1 (1; 4-beta-D-glucan cellobiohydrolase) promotor of gene C BH1 is an extremely strong inducible promoter, and this promotor can be by substrate for induction such as lactose, wheat straw powder and Mierocrystalline celluloses, but can be checked by glucose.Simultaneously, it also can be used as the homologous nucleotide sequence of exogenous origin gene integrator to the mould genome of wood.The mould method for transformation of Rui Shi wood mainly contains electro fusion method and protoplast transformation method etc., the screening of recon mainly be through the employing antibiotics resistance as selected marker gene, the most frequently used is Totomycin and G418 resistant gene.
Rui Shi wood is mould ripe not enough as a kind of new its operative technique of foreign protein genes expression system, and fragmentary successful report (seeing table 1) is only arranged at present.
The external source recombinant protein of the mould expression of table 1 Rui Shi wood
| Title | The source | Molecular weight (kDa) | Promotor | The host | Expression amount | Reference |
| Ethylene synthetase | Pseudomonas?syringae pv.glycinea | 40 | Cbh1 | T.viride | ? | Li?Tao?et al.，2008 |
| Zytase | Acrophialophora nainiana | 19 | Cbh1 | T.reesei | 172mg/L | Bruno?C，et al.，2007 |
| Esterase | Melanocarpus?albomyces | 74 | Cbh1 | T.reesei | ? | Hanna?K.，et al.，2005 |
| EAFP | Aspergillus? |
6 | Cbh1 | T.viride | ? | Xu Jun etc., 2003 |
| Barley endopeptidase B | Barley | 30 | Cbh1 | T.reesei | 500mg/L | Ritva?S.，et al.，1997 |
Before the present invention; The report of do not adopt also that codon is optimized, the T4 lysozyme gene of synthetic being expressed this foreign protein with the induction type mode in Rui Shi wood is mould; Also nobody uses the promotor of the mould cellobiohydrolase 1 gene C BH1 of Rui Shi wood as homologous sequence the T4 lysozyme gene to be incorporated into the report in the mould genome of Rui Shi wood, more has no talent and in Rui Shi wood is mould, carries out the high density fermentation production of T4 N,O-Diacetylmuramidase recombinant protein.
Summary of the invention
First purpose of the present invention is to provide that a kind of codon is optimized, the T4 lysozyme gene of synthetic, and it can obtain stable in Rui Shi wood is mould and efficiently express.
Second purpose of the present invention is to use one with the exogenous nucleotide integration sequence; The plasmid vector of important Expression elements such as carrying T4 N,O-Diacetylmuramidase foreign gene and selection markers can be integrated in the mould genome of Rui Shi wood, and obtain the mould engineering strain of reorganization Rui Shi wood of genetic stability.
The 3rd purpose of the present invention is that a kind of mould reorganization bacterium of Rui Shi wood that under mould cellobiohydrolase 1 gene promoter of Rui Shi wood starts, stably efficiently expresses the T4 lysozyme gene will be provided.
The 4th purpose of the present invention provides the righttest reorganization Rui Shi mould growth of wood and expression condition and target protein purification process fast.
For achieving the above object, the present invention at first provides a kind of gene of the T4 of coding antalzyme protein, and it is according to the gene codon optimized mistake of the mould preference of Rui Shi wood, that encode or at least partly encode the T4 antalzyme protein.Wherein said T4 antalzyme protein primary structure is the aminoacid sequence shown in the SEQ ID NO.1.According to an embodiment preferred, said gene has or at least partly has the nucleotide sequence shown in the SEQ ID NO.2.
Said then gene is placed under the mould cellobiohydrolase 1 gene promoter manipulation of Rui Shi wood, is incorporated in the mould genome of Rui Shi wood, and along with the growth of reorganization Trichoderma body, the T4 lysozyme gene can stably be efficiently expressed with the induction type mode.
According to an embodiment preferred; The promotor of cellobiohydrolase 1 gene that Rui Shi wood is mould and terminator dna sequence dna are successively cloned out and are inserted in the new mould expression plasmid carrier of T4 N,O-Diacetylmuramidase wood; By the homologous recombination that takes place between this promotor or terminator dna sequence dna and the mould genome of Rui Shi wood; Can the T4 lysozyme gene that receive the mould cellobiohydrolase 1 gene promoter regulation and control of Rui Shi wood and other important Expression element be incorporated in the mould genome of Rui Shi wood simultaneously; And the mould engineering strain of Rui Shi wood that obtains recombinating; It can be stablized under cellulosic inducing and express T4 N,O-Diacetylmuramidase recombinant protein efficiently, and it is secreted into outside the born of the same parents the most at last.
Concrete; The gene that at first is coding T4 N,O-Diacetylmuramidase is come out according to the complete synthetic of the Rui Shi mould codon of having a preference for of wood; Then through the pcr amplification technology; In the promotor that its 5 ' end upstream region adds the nucleotide coding sequence and mould cellobiohydrolase 1 gene of Rui Shi wood of mould cellobiohydrolase 1 secreting signal peptide of Rui Shi wood, the terminator sequence in that its 3 ' end downstream area adds mould cellobiohydrolase 1 gene of Rui Shi wood forms a new foreign gene reading frame thus.According to an embodiment preferred, the T4 lysozyme gene at first is placed under the inducible promoter regulation and control, is inserted into then on the mould expression plasmid carrier of Rui Shi wood.On this expression plasmid carrier; This inducible promoter is positioned at the upper reaches of cellobiohydrolase 1 secreting signal peptide nucleotide coding sequence and the formed fusion gene of T4 N,O-Diacetylmuramidase nucleotide coding sequence, and it handles the stable and efficiently expression of so-called antigen-4 fusion protein gene in Rui Shi wood is mould.Simultaneously, this promotor nucleotides sequence is listed in the effect that the expression plasmid vector integration has also played the homologous recombination sequence in the mould genomic process of Rui Shi wood.At this expression plasmid carrier after complete linearization process; Can be directed in the mould cell of Rui Shi wood through methods such as electro fusion method or protoplastiss; And then passing through the homologous recombination stable integration on the mould genome group of Rui Shi wood, the mould T4 N,O-Diacetylmuramidase recombinant protein of in the fermentation culture process, expressing of this reorganization Rui Shi wood is able to be secreted in the substratum outside the born of the same parents under the guiding of cellobiohydrolase 1 secreting signal peptide.
The recombinant protein of indication of the present invention can be the whole of T4 antalzyme protein, yet in some specific embodiments, expressed foreign gene also possibly be the part of this native protein.Sometimes, the protein gene that T4 N,O-Diacetylmuramidase recombinant protein can have a different biological function with another one, its objective is and purifies for ease or improve its biological activity simultaneously at a mould cell inner expression of wood with independence or amalgamation mode.Proteic fusion can be passed through protein translation post-treatment, covalently bound mode, perhaps through the DNA recombinant technology gene just is stitched together each other before accurate translation, and these two kinds of technology is technology that those skilled in the art know already.
Like this, the present invention more preferably embodiment is: 1) according to the mould codon of having a preference for of Rui Shi wood, carry out the synthetic of T4 lysozyme gene; 2) clone of Rui Shi mould cellobiohydrolase 1 gene promoter of wood and cellobiohydrolase 1 secreting signal peptide nucleotide coding sequence; 3) clone of cellobiohydrolase 1 gene terminator DNA; 4) make up a plasmid expression vector; The gene (comprise that gene codon is optimised, change or merge mutually etc.) that wherein contains the gene or derivatives thereof of coding T4 N,O-Diacetylmuramidase recombinant protein with other gene; This gene at first is spliced to form fusion gene with the mould cellobiohydrolase 1 secreting signal peptide nucleotide coding sequence of Rui Shi wood each other; Be placed in then under the mould cellobiohydrolase 1 gene promoter control of Rui Shi wood; Be added with cellobiohydrolase 1 terminator dna sequence dna in these fusion gene downstream; Also include in the plasmid expression vector simultaneously and be used for exogenous origin gene integrator to the mould genomic homologous recombination sequence of Rui Shi wood, this sequence can be Rui Shi mould cellobiohydrolase 1 gene promoter of wood or other homologous sequences; 5) through electro fusion method or additive method; Plasmid expression vector DNA after the above-mentioned linearizing is transformed in the mould protoplastis of Rui Shi wood; And the mould protoplastis of the Rui Shi after will transforming wood coats on the HM solid plate substratum that contains 0.5mg/mL G418 and grows, and the wooden mould single bacterium colony that can on this substratum, grow is exactly the wooden mould recon that contains foreign gene.
Further; The invention provides about reorganization reorganization Rui Shi wood the righttest mould grown cultures condition and exogenous protein expression condition; Composition, inoculum size, pH value, dissolved oxygen amount and incubation time etc. like substratum make the increment of this wooden mould engineering bacteria of recombinating and the secretory volume of recombinant protein all reach maximization as much as possible thus.From fermented liquid, can obtain highly purified T4 N,O-Diacetylmuramidase recombinant protein through operations such as bactofugation, tubular fibre filtration, ion exchange chromatographies, for suitability for industrialized production, low-cost fermentation and the fast purifying of this enzyme lay the foundation later on.Specifically comprise following three phases: in 1) the yeast culture stage, behind 10% the mould engineering bacteria of inoculum size inoculation Rui Shi wood, through 72 hours cultivation, Trichoderma body weight in wet base will reach about 35～40g/L; 2) the abduction delivering stage adds Mierocrystalline cellulose, induces wooden mould cell high-efficient to express T4 N,O-Diacetylmuramidase recombinant protein and it is secreted in the substratum, keeps certain pH value and dissolved oxygen amount; 3) protein purification, fermented liquid is handled through the membrane filtration of centrifugal and three different sizes, and at process primary ions displacement chromatography, the purity of T4 antalzyme protein can reach more than 99%.
Most preferred; Adopt the method for engineering strain fermentative prodn recombinant protein of the present invention to comprise: (1) seed liquor preparation: at first with mycelium inoculation on the PDA substratum; Cultivated 6～7 days for 27～28 ℃; Again spore is dissolved in the sterilized water, is inoculated in the liquid MM substratum, 27～28 ℃ of shaking culture (375～380 rev/mins) 72～96 hours with 1～2% volume ratio; With its seed liquor as high density fermentation;
(2) yeast culture: in fermentor tank, hold an amount of MM basis fermention medium; The wooden mould seed liquor of volume ratio inoculation step (1) by 8～10%; 27～28 ℃ of aeration-agitations (rotating speed maintains 375～380 rev/mins from start to finish) were cultivated about 72～96 hours, and pH maintains about 5.5～6;
(3) protein induced expression: at postvaccinal the 4th～6 day; Stream adds the Mierocrystalline cellulose that is dissolved in 50wt% in the MM substratum in the above-mentioned substratum; The substratum cellulose concentration is maintained about 2～3wt%, and dissolved oxygen amount is greater than 20%, and temperature maintenance is at 27～28 ℃; PH maintains about 5.5～6, under this condition, continues vibration (rotating speed maintains 375～380 rev/mins from start to finish) inducing culture 3～4 days.
The MM culture medium prescription of in said process, using is: every liter contains: glucose 20g, (NH
4)
2SO
45g, KH
2PO
415g, MgSO
47H
2O 0.6g, CaCl
20.6g, FeSO
47H
2O 0.005g, MnSO
4H
2O 0.0016g, ZnSO
47H
2O 0.0014g, CoCl
20.002g pH 5.5.
The purity of T4 N,O-Diacetylmuramidase recombinant protein is directly connected to the height of this Application of Recombinant scope and the related prods cost of producing in the leavened prod; According to a preferential embodiment; For a large amount of T4 N,O-Diacetylmuramidase recombinant protein of fast purifying; Can be at first use filtration unit and the ion exchange chromatography etc. of the different molecular weight that dam to carry out pre-treatment the mould fermented liquid supernatant liquid of Rui Shi wood; Each link in purge process all can use polyacrylamide gel electrophoresis to detect, and can obtain purity through aforesaid method and reach 99% recombinant protein.
The present invention has only mentioned Rui Shi trichoderma strain QM9414 when introducing the mould expression system of Rui Shi wood in detail; Yet; As the wooden mould expression system that those skilled in the art knew already, the wooden mould expression system of many kinds can utilize method provided by the present invention to carry out genetic transformation, expression and production.Therefore, all these wooden mould expression systems all should be included within the claim scope of the present invention.
Just as in the following instance that kind that will describe in detail; Used plasmid vector is an integrated plasmid expression system in the mould method for transformation of Rui Shi wood that the present invention describes; According to a preferential embodiment; Plasmid expression vector used in the present invention is one and obtains improved pPIC9K; Its original AOX1 inducible promoter is replaced by the mould cellobiohydrolase 1 gene induced type promotor of Rui Shi wood, also includes in the plasmid expression vector simultaneously and is used for the homologous recombination sequence of exogenous origin gene integrator to the yeast genes group, and this sequence can be the mould cellobiohydrolase 1 gene induced type promotor of Rui Shi wood, terminator sequence or other homologous sequence.
The present invention has greatly improved the method for T4 N,O-Diacetylmuramidase recombinant protein biological yield in the mould cell of Rui Shi wood, and this recombinant protein is known to have very strong antibacterial or germicidal action under native state; Or or rather; The T4 N,O-Diacetylmuramidase recombinant protein of indication of the present invention is similar with this recombinant protein that the albumen that is derived from T4 phage gp e coded by said gene or other conventional expression system are produced, and can be widely applied to fields such as medical treatment, food, feed and scientific research as antibacterial or sterilant.Adopt method of the present invention; Can be effectively codon optimized T4 lysozyme gene be incorporated in the mould genome of Rui Shi wood through Rui Shi mould cellobiohydrolase 1 gene promoter of wood or the terminator sequence mode with homologous recombination; The mould engineering strain of Rui Shi wood that obtains can with induction type mode formula stable with express T4 N,O-Diacetylmuramidase recombinant protein efficiently; The fermentation and the purifying process of this recombinant protein are provided simultaneously, have been applicable to large-scale production T4 N,O-Diacetylmuramidase recombinant protein.
Description of drawings
Fig. 1 is the codon optimized and transformation front and back contrast of T4 lysozyme gene, the gene order that the N representative is codon optimized and transformation is preceding; M represents codon optimized and improved gene order, has the expression nucleotide base of ordinate not change between two sequences;
Fig. 2 is the building process of the mould efficient expression vector T4-CBH9K of induction type wood;
Fig. 3 is that the pcr amplification of wooden mould conversion recon is identified: 1 is the DNA standard molecular weight; 2～9 is the T4 lysozyme gene amplification of 9 mould transformants of wood; CK+ is a T4 lysozyme gene positive control; CK-is a T4 lysozyme gene negative control;
Fig. 4 is standard molecular weight albumen (Blue Plus Protein marker 12～94KDa, Beijing TransGenBiotech Company products) for SDS-PAGE electrophoresis detection T4 N,O-Diacetylmuramidase expression of recombinant proteins situation: M; The negative contrast of CK-, 120 hours fermented liquid supernatant of (not containing the T4 lysozyme gene) reorganization trichoderma strain inducing culture that CBH9K transforms; 1 cultivates 72 hours fermented supernatant fluid before inducing for TR-T4; 2～4 are respectively the fermented supernatant fluid of TR-T4 inducing culture after 24,48 and 72 hours;
Fig. 5 measures for reorganization T4 lysozyme activity: X-coordinate is the pure article of reorganization T4 N,O-Diacetylmuramidase (unit is mg/mL) of different weaker concns; Ordinate zou is the OD350nm absorbance value; T4lysozyme is the determination of activity result of the pure article of T4 N,O-Diacetylmuramidase that in wooden mould engineering bacteria TR-T4 fermented liquid, prepare, and CK-is the determination of activity result that CBK9K (empty carrier) transforms the different weaker concns of Trichoderma fermented supernatant fluid extract.
Embodiment
Below in conjunction with accompanying drawing and embodiment, specific embodiments of the invention describes in further detail.Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
Embodiment 1: the synthetic of the T4 lysozyme gene after codon optimized
The T4 lysozyme gene derives from the T4 phage; Its codon and prokaryotic organism are more approaching; And the Rui Shi Trichoderma is in eukaryote; Therefore they are existing certain difference aspect the gene codon preference, and this species diversity has influence on T4 lysozyme gene and the stability and the expression efficiency of transcription product in the mould cell of Rui Shi wood thereof possibly.For improving the biological yield of T4 N,O-Diacetylmuramidase recombinant protein; Dna nucleotide sequence and aminoacid sequence (GenBank:NC000866) according to the T4 lysozyme gene of having announced already; Under the prerequisite that does not change its aminoacid sequence, (see SEQ ID NO.1); According to the Rui Shi wood mould codon of having a preference for (seeing CodonUsageDatabase:Hypocrea jecorina), artificial design and the dna encoding sequence of having synthesized new T4 N,O-Diacetylmuramidase maturation protein.T4 N,O-Diacetylmuramidase mature protein gene after codon optimized with transform before compare, changed 159 nucleotide bases wherein, relate to 53 codons altogether, and the content of G+C becomes 58.8% (seeing SEQID NO.2) by original 36.6%.Accompanying drawing 1 (said gene splicing with synthetic work by Shanghai Bo Ya biotech company on behalf of completion) is seen in contrast before and after the genetic modification.
Embodiment 2: the clone of mould cellobiohydrolase 1 gene promoter of Rui Shi wood-secreting signal peptide and terminator nucleotides sequence
1, the mould genomic extraction of Rui Shi wood
Rui Shi trichoderma strain QM9414 (this bacterial strain is from China Agricultural University) is seeded in MM, and [every liter contains glucose 20g, (NH
4)
2SO
45g, KH
2PO
415g, MgSO
47H
2O0.6g, CaCl
20.6g, FeSO
47H
2O 0.005g, MnSO
4H
2O 0.0016g, ZnSO
47H
2O 0.0014g, CoCl
20.002g, 2% (w/v) agar powder, pH 5.5] on the solid medium, cultivated 4～5 days for 28 ℃; The a small amount of mycelium of picking is transferred to the centrifuge tube from flat board, washes 2 times with saline water, adds a small amount of granulated glass sphere and 500 μ lL STES damping fluid [200mM TrisHCl (pH8.5) again; 250mM NaCl, 25mM EDTA, 2%SDS]; Behind the 3 * 40s that vibrates on the vortex vibrator, 65 ℃ of reaction 10min, ice bath 5min.After adding 200 μ L 10M ammonium acetate ice bath 5min again, the centrifugal 10min of 12000rpm.Get supernatant, add 200 μ L 10M ammonium acetates ice bath 5min once more, the centrifugal 10min of 12000rpm.With supernatant with Virahol-20 ℃ deposition 20min.The centrifugal 10min of 12000rpm, deposition is with 70% washing with alcohol twice, and oven dry with 10 μ L water dissolution, adds an amount of RNase A, and behind 37 ℃ of reaction 30min ,-20 ℃ are subsequent use.
2, the clone of mould cellobiohydrolase 1 gene promoter of Rui Shi wood-signal peptide and terminator nucleotides sequence
According to known Rui Shi wood mould cellobiose hydrolase gene promotor-secreting signal peptide (GenBank:D86235 and E00389) and terminator sequence (GenBank:E00389); Designed primer1, primer2, primer3 and 4 primers of primer4 respectively, wherein primer Primer1 sequence is (seeing SEQ ID NO.3): 5 '-AC
AATTCTGGAGACGGCTTGTTGA ATC-3 ', Primer2 are (seeing SEQ ID NO.4): 5 '-
CTCGAAGATGTTCATAGCACGAGCTGTGGCCAAGAAGGC-3 ', Primer3 are (seeing SEQ ID NO.5): 5 '-
TACAAGAACTTGTAAAGGTCACCTTCTCCAACATCAAGTTCG-3 ', Primer4 are (seeing SEQ ID NO.6): 5 '-AC
CACGAAGAGCGGCGATTCTACGGG-3 '.Following stroke wavy line is partly represented restriction enzyme digestion sites in the primer, and following stroke horizontal line partly is the part that is base complementrity with T4 lysozyme gene two terminal preceding 15 nucleotide base sequences of synthetic respectively.With Primer1 and Primer2 is primer; With Rui Shi trichoderma strain QM9414 genomic dna is template; Through pcr amplification; Amplification obtains mould cellobiohydrolase 1 gene promoter of Rui Shi wood-secreting signal peptide nucleotide coding sequence (Pcbh1-sig) (annotate: cellobiohydrolase 1 secreting signal peptide length is 17 amino-acid residues, and is coded by 51 nucleotide bases), is the structure of convenient follow-up expression vector; In Primer1, added Bgl II restriction enzyme digestion sites (seeing following stroke of wavy line part among the Primer1); In Primer2, added the dna sequence dna (seeing the part that following stroke horizontal line is arranged among the Primer2) that is base complementrity with preceding 15 nucleotide bases of T4 N,O-Diacetylmuramidase nucleotide coding sequence 5 ' end; The pcr amplification condition is: 94 ℃ of preparatory sex change 5 minutes, and 94 ℃ of sex change 40 seconds, 55 ℃ of annealing 40 seconds, 72 ℃ were extended 1.5 minutes, 30 circulations, last 72 ℃ were extended 10 minutes.Likewise, be primer with Primer3 and Primer4, be template with Rui Shi trichoderma strain QM9414 genomic dna, through pcr amplification, obtained the mould cellobiohydrolase 1 gene terminator sequence (Tcbh1) of Rui Shi wood; Structure for convenient follow-up expression vector in Primer3, has added the dna sequence dna (seeing the part that following stroke horizontal line is arranged among the Primer3) that is base complementrity with preceding 15 nucleotide bases of T4 N,O-Diacetylmuramidase nucleotide coding sequence 3 ' end; In Primer4, added Sal I restriction enzyme digestion sites (seeing following stroke of wavy line part among the Primer4).The pcr amplification condition is: 94 ℃ of preparatory sex change 5 minutes, and 94 ℃ of sex change 40 seconds, 55 ℃ of annealing 40 seconds, 72 ℃ were extended 45 seconds, 30 circulations, last 72 ℃ were extended 10 minutes.
The splicing of the mould gene reading frame of embodiment 3:T4 N,O-Diacetylmuramidase Rui Shi wood
The PCR product of above-mentioned codon optimized T4 lysozyme gene (artificial sequence), Pcbh1-sig and Tcbh1 is carried out agarose gel electrophoresis, use gel to reclaim test kit (Beijing hundred Tyke Bioisystech Co., Ltd, method is seen this description of product) and reclaim target DNA fragment; According to 1: 1: 1 ratio, Pcbh1-sig, T4 lysozyme gene and three kinds of dna fragmentations of Tcbh1 (gel recovery product) are mixed, as template; Carrying out PCR increases again; Used two ends primer is respectively above-mentioned primer1 and primer4, and the pcr amplification condition is: 94 ℃ of preparatory sex change 5 minutes, 94 ℃ of sex change 40 seconds; Annealed 40 seconds for 55 ℃; 72 ℃ were extended 2 minutes, 30 circulations, and last 72 ℃ were extended 10 minutes.
The PCR product of above-mentioned total length directly is inserted into pEASY-T1 (available from the Beijing Quanshijin Biotechnology Co., Ltd; Working method is seen this product description) in the T site in the plasmid; According to the method that the said firm provided; The middle plasmid vector T4-T that obtains containing the exogenous dna fragment insertion (contains the mould gene reading frame of T4 N,O-Diacetylmuramidase Rui Shi wood; It is in proper order: the bacterial clone T4 lysozyme gene-cellobiohydrolase 1 gene terminator sequence behind cellobiohydrolase 1 gene promoter-cellobiohydrolase 1 secretion signal peptide-coding sequence-codon optimized); Then, through the nucleotide sequencing analysis, confirm that the dna sequence dna in the mould gene reading frame of T4 N,O-Diacetylmuramidase Rui Shi wood is correct and complete (dna sequencing is accomplished by Beijing Biokit, Inc.).
Embodiment 4: the structure of the mould expression vector T4-CBH9K of Rui Shi wood
Carry out double digestion with restriction enzyme Bgl II and Sal I, the mould gene reading frame of the wood of the T4 N,O-Diacetylmuramidase Rui Shi in the plasmid vector T4-T in the middle of being connected is scaled off, utilize agarose gel electrophoresis to separate the purpose fragment.Handle plasmid pPIC9K with identical restriction enzyme, utilize agarose gel electrophoresis to separate the purpose fragment.Just obtain Rui Shi wood mould expression vector T4-CBH9K (seeing accompanying drawing 2) with linking together after the dna fragmentation mixing of above-mentioned two recovery and with ligase enzyme, use above-mentioned plasmid vector transformed into escherichia coli cell DH5 α (available from U.S. GIBCO company) then conveniently to carry out duplicating and preserving of this plasmid.
Plasmid vector pPIC9K is available from American I nvitrogen company; It is a yeast inducible expression plasmid vector; It contains an inducible promoter-alcohol oxidase promotor (AOX1) efficiently, and under the inducing of methyl alcohol, efficiently expressing of foreign gene inserted in adjustable downstream.
Embodiment 5: the preparation of linearizing T4-CBH9K DNA
At first use alkaline lysis (referring to the molecular cloning test guide) from Bacillus coli cells DH5 α, to extract the T4-CBH9K DNA; Cut with 1～2 times of excessive restriction enzyme Nde I enzyme then; Make it complete linearizing, utilize agarose gel electrophoresis to detect enzyme and cut whether fully.Then with phenol and chloroform respectively the above-mentioned enzyme of extracting cut product, ethanol sedimentation is abandoned supernatant, collecting precipitation after lyophilize, is dissolved in deposition in the aseptic deionized water again ,-20 ℃ of preservations are subsequent use.
Embodiment 6: the genetic transformation of the mould protoplastis of Rui Shi wood
The conidium that on PDA substratum (agar 15g adds water to 1L for peeling yam 200g, glucose 20g) inclined-plane, cultivate about 7 days the mould QM9414 of Rui Shi wood washes with saline water.On the PDA plate culture medium, place an aseptic zellglas, with Rui Shi trichoderma conidium suspension (concentration be about 107/mL) coat on the substratum, cultivate about 18h for 28 ℃.20mg/mL lywallzyme (available from the Guangdong Microbes Inst), 20mg/mL Snailase and 10mg/mL cellulase are mixed; Be dissolved in fully in 1.2M sorbyl alcohol～100mM potassium phosphate buffer (pH5.6); Again the young tender mycelia on the zellglas in the flat board is transferred to wherein 28 ℃ of water-bath 1.5h～2h.Microscopy frequently in this process, the generation situation of observing protoplastis.Filter through 6 layers of lens wiping paper, protoplastis and the mycelium that is not digested are separated, and with 1.2M sorbyl alcohol～10mMTrisHCl (pH7.5) washing 2 times.6000 rev/mins centrifugal 10 minutes, collect protoplastis, and wash 2 times with 1M sorb～10mMTrisHCI (pH7.5), be resuspended at last among 1M sorbyl alcohol～10mMCaCl2-10mM TrisHCl (pH7.5), making protoplastis concentration is 5x10
7-5x10
8/ mL.Linear recombinant plasmid (2～10 μ g) is mixed with the Rui Shi mould protoplastis of wood (about 200 μ L) for preparing, add 200 μ L 60%PEG4000～50mMCaCl
2～10mMTrisHCl (pH7.5), ice bath 30min, the room temperature temperature is bathed 20min then, adds 2mL 60% PEG4000～50mM CaCl again
2～10mmol/LTrisHCl (pH7.5) continues at room temperature and places 5min, adds 4mL 1M sorbyl alcohol～10mmol/LCaCl at last
2～10mmol/LTrisHCl (pH7.5), mixing is coated on above-mentioned conversion fluid and contains the MH that G418 concentration is 0.5mg/mL [every liter contains dextrose 20g, (NH
4)
2SO
45g, KH
2PO
415g, MgSO
47H
2O 0.6g, CaCl
20.6g, FeSO
47H
2O 0.005g, MnSO
4H
2O0.0016g, ZnSO
47H
2O 0.0014g, CoCl
20.002g, sorbitol 182.18g, pH5.5] on the flat board, cultivate for 28 ℃ and grew to wooden mould transformant in 4～5 days.
Embodiment 7: the screening of reorganization Rui Shi trichoderma strain
The G418 resistance transformant of growing on the MH substratum is seeded in respectively on the MM culture medium flat plate, cultivates after 4～5 days for 28 ℃, a small amount of mycelium of picking is transferred to the different centrifuge tubes respectively from flat board; Wash 2 times with saline water, add a small amount of granulated glass sphere and 500 μ LSTES damping fluid [200mM TrisHCl (pH8.5), 250mM NaCl again; 25mM EDTA, 2%SDS], behind the 3 * 40s that vibrates on the vibrator; 65 ℃ were reacted ice bath 5 minutes 10 minutes.Add 200 μ L 10M ammonium acetate solutions again, ice bath is after 5 minutes, 12000 rev/mins centrifugal 10 minutes, get supernatant, add 200 μ L 10M ammonium acetate solutions, ice bath is 5 minutes once more, 12000 rev/mins centrifugal 10 minutes.Get supernatant and with Virahol-20 ℃ deposition 20 minutes, centrifugal then, deposition is with 70% washing with alcohol twice, air-dry after, with 10 μ L water dissolution, add an amount of RNase A, 37 ℃ were reacted after 30 minutes, subsequent use.
The mould genomic dna of Rui Shi wood with said extracted is a template; With P1 (seeing SEQ IDNO.7) (5 '-AAATGA ACATCTTCGAGAAGT TGAGG-3 ') and P2 (seeing SEQ ID NO.8) (5 '-AATTACAAGTTCTTGTAGGCGTCCC-3 ') is primer; Through PCR, the T4 lysozyme gene of amplification 495bp.Reaction conditions is: 95 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 40s, totally 30 circulations, 72 ℃ are extended 10min, select clone's (seeing the swimming lane 7 in the accompanying drawing 3) of positive band.
To there be the Trichoderma silk of positive band to be seeded in once more on the PDA inclined-plane, cultivate about a week for 28 ℃, make its a large amount of generation conidiums.With saline water conidium is washed earlier, filter with 12 layers of lens wiping paper then, to remove unnecessary mycelia.Trichoderma conidium is coated on the thin PDA culture medium flat plate according to 100 spore/wares uniformly; Cultivate about 10h for 28 ℃; In super clean bench, observe the spore germination situation on the plate culture medium with 10 times of object lens of microscope, with sterile razor blade with in the visual field independently and the conidium that sprouts together downcut together with substratum.Change on the PDA inclined-plane that contains 0.4mg/mL G418, cultivate about a week for 28 ℃, when in vitro having a large amount of mycelia to produce, it is chosen a part, the rapid extraction genome carries out the pcr amplification checking and whether still contains the T4 lysozyme gene, and amplification condition is the same.Still positive clone through after above-mentioned monospore separation and the PCR detection is confirmed to be the wooden mould transformant of success, and is named as TR-T4.
Embodiment 8: the high density fermentation of the wooden mould engineering bacteria of recombinating
1, the preparation of seed liquor
With the mycelium inoculation of engineering strain TR-T4 in a plurality of PDA solid slant culture bases; Cultivated 6～7 days for 27～28 ℃; After treating that conidium grows up to green, scrape spore and be suspended in the sterilized water, be inoculated in the 500mL MM liquid nutrient medium with 1～2% inoculum size; 48～72h is cultivated in 27～28 ℃ of lucifuge concussions (375～380 rev/mins), with its seed liquor as high density fermentation.
2, the mould high density fermentation in the 5L fermentor tank of reorganization wood
This fermenting process can be divided into following two stages: 1) the yeast culture stage: in 5L fermentor tank (Zhenjiang Oriental Bio-engineering Technology Co., Ltd), held 3.5L MM basis fermention medium; The wooden mould seed liquor that ratio inoculation in 10% prepares before this; 27～28 ℃ of aeration-agitations (rotating speed maintains 375～380 rev/mins from start to finish) were cultivated about 72～96 hours, and pH maintains about 5.5～6.In this culturing process, along with the growth of Trichoderma body, the dissolved oxygen amount in the substratum will reduce by 100% gradually, and after the carbon source in the substratum runs out of basically, dissolved oxygen amount will be increased to more than 80% once again, and the weight in wet base of thalline will reach 35～40g/L this moment.2) the abduction delivering stage (72～144h): after inoculation the 4th day; Add 50% cellulose suspension (with the preparation of MM substratum) through peristaltic pump stream; Make its working concentration maintain about 2～3% all the time; Dissolved oxygen amount is cultured to inoculation back 144～166 hours greater than 20%, 27～28 ℃ of aeration-agitations all the time, and pH maintains about 5.5～6.Whenever descended centrifugal 10 minutes for 4 ℃ through 5000rpm at a distance from 24 hours peek milliliter fermented liquids; Get 30uL fermented liquid supernatant liquid and carry out the SDS-PAGE detection; Discovery has the observable protein band of naked eyes, and molecular weight is about 19KDa, matches with the molecular weight of T4 N,O-Diacetylmuramidase recombinant protein in inferring.In addition, see that adding the Mierocrystalline cellulose inducing culture after 72 hours, the Recombinant Protein Expression amount reaches climax (seeing accompanying drawing 4) from the PAGE electrophorogram.
Embodiment 9: the purifying of recombinant protein
After treating that a fermentation period is all over, leave and take the 400mL fermented liquid directly carries out next round as seed liquor (inoculum size is 10%) fermenting process.Similar operation adds up to carry out 3 takes turns, and takes turns in the fermenting process every, all the increment and the T4 N,O-Diacetylmuramidase Recombinant Protein Expression amount of thalline is measured.In addition, take turns after fermenting process finishes fully, also get a little bacterium liquid and coat on the PDA solid plate every; And therefrom any mould single bacterium colony of 10 wood of picking, its genomic dna of rapid extraction carries out PCR and detects; The result finds; The living weight of thalline, the speed of growth and Recombinant Protein Expression amount are taken turns kept stable in the fermenting process at each, and in addition, the detected result of PCR also confirms the wooden mould good genetic stability (seeing table 2) that has of reorganization.
The genetic stability of table 2TR-T4 bacterial strain and the mensuration of exogenous protein expression stability
| PCR is positive | The thalline weight in wet base (g/L) of growing 72 hours | Induce 72 hours T4 N,O-Diacetylmuramidase expression amounts (mg/L) |
| ?100％ | 39 | 550 |
| ?100％ | 37 | 540 |
| ?100％ | 40 | 590 |
Except that the seed liquor of retaining, remaining fermented liquid is used for the purifying of T4 N,O-Diacetylmuramidase recombinant protein.Fermented liquid is left and taken supernatant through centrifugal 10 minutes of 4 ℃ of 5000rpm.Fermented liquid supernatant is at first used tubular fibre filter post (Tianjin MoTian Membrane Engineering Technology Co., Ltd, product type: MOF-503 method of use the see the said firm explanation) filtration of molecular weight as 50KDa of damming; Collect and see through liquid; The clarifying liquid that sees through uses the molecular weight that dams to handle as the nf membrane of 10KDa (the bright utmost point in Shanghai Chemical Industry Science Co., Ltd, PIN: 2426538, method of use is seen the said firm's explanation) again; Keep phegma, final volume is about 700mL.This phegma is carried out desalting treatment; In the 700mL phegma, add 6.3L zero(ppm) water; Be about to 10 times of phegma dilutions, then with diluent once more through the nanofiltration membrane treatment of above-mentioned 10KDa, the also corresponding dilution of salt ionic concentration is 10 times in the phegma that obtains at this moment; So repetitive operation is 5 times, i.e. salt ionic concentration dilution 10 in the phegma
5Doubly, finally obtain the 700mL phegma, after this phegma lyophilize, can obtain the recombinant protein lyophilized powder of preliminary purification (purity is big by about 60%).
This albumen lyophilized powder sample can obtain further purifying through ion exchange chromatography, to satisfy the different production demand.Employed cation ion exchange resin is CM-Sephadex-C25 (U.S. GE Company products).According to the said firm's description of product, resin is carried out pre-treatment, adorn post (the dress column method is seen the description of product, and chromatograph is Shanghai Hu Xi analytical instrument Co., Ltd., Factory product-model MA99-3) then, concrete operation method is following
1) zero(ppm) water of 5 times of volumes of albumen lyophilized powder adding dilutes, and with third acetic acid this diluent pH value is transferred to 5.5～5.8 then.
2) sample upper prop (annotate: when being 2g+500mL zero(ppm) water as if the lyophilized powder sample, use the chromatography column of 1.5x50cm, the resin bed volume height is 40cm), flow velocity 3mL/ minute.Go out process with ultraviolet Protein Detection appearance and registering instrument record eluent stream.
3) wash post, after treating that sample is fast and all getting into resins, contain 10 with the 0.1M phosphate buffered saline buffer
-3M MgSO
4(pH6.5) wash post, flow velocity 3mL/ minute is zero to the OD280nm light absorption value, and needing damping fluid approximately is 5～6 times of column volumes.
4) wash-out contains 10 with the 0.1M phosphate buffered saline buffer
-3M MgSO
4Carry out the target protein wash-out with 0.5M NaCl (pH6.5); Flow velocity 3mL/ minute; Keep watch on the protein stream artificial situation and collect maximum albumen wash-out part (target protein peak generally appear at add behind the elutriant soon) with ultraviolet Protein Detection appearance, collect the elutriant that contains target protein.
5) with in the elutriant dislocation dialysis tubing, zero(ppm) water is carried out dialysed overnight, during change zero(ppm) water for several times, purpose is in order to remove wherein contained NaCl.
6) dialyzate can obtain target protein dry powder through lyophilize, and purity of protein can reach 99% (SDS-PAGE electrophorogram through the LabWork software analysis, U.S. UVP Company products).More than, but prolonged preservation under the room temperature.Look the application target difference, can be processed into various formulations, like foodstuff additive, injection etc.
The bioactive detection of embodiment 9:T4 N,O-Diacetylmuramidase recombinant protein
Utilize colourimetry to carry out the bacteriolyze specific activity, concrete operations are following:
1. substrate preparation: from-72 ℃ of Ultralow Temperature Freezers, take out the micrococcus (Micrococcus Lysodeikticus) (available from Chinese common micro-organisms DSMZ, bacterium numbering 1.634) of freezing, behind a little bacterium liquid of inoculating needle picking; At LB (5g yeast extract, 10g Tryptones, 10g NaCl; 20g agar; Add water to 1L, pH7.0) rule on the solid plate substratum, be inverted overnight cultures for 37 ℃.
The single bacterium colony of picking micrococcus cell cultures is inoculated in the 5mL LB liquid nutrient medium, and 37 ℃ of shaking culture are spent the night, and all transfer next day in 100mL LB liquid nutrient medium, and 37 ℃ of shaking culture are to cell concn OD
600nmValue is 1.602.Nutrient solution is transferred in the 100mL centrifuge tube, and 4 ℃ of 5000rpm centrifugal 10 minutes behind the ice bath, collecting precipitation.Suspend and washed cell with 0.05M Tris-HCl (pH7.4) damping fluid 50mL, the same centrifugal after, collecting precipitation.Cell is suspended in the identical Tris-HCl damping fluid of 10mL once more, and changes and to be displaced in the drying bottle, place-20 ℃ of freezing 1 weeks (refrigerated storage is essential to strengthening the cell susceptibility), and then carry out lyophilize.Cell dry powder can place prolonged preservation under the moisture eliminator room temperature in confined conditions.If cell dry powder is insensitive to the T4 N,O-Diacetylmuramidase, also can place 37 ℃ to preserve in cell dry powder so that strengthen the susceptibility of cell.
Get micrococcus lyophilized powder 40mg, be suspended in phosphate buffered saline buffer (the 5.54g Na of 100mL 0.2M again
2HPO
412H
2O, 0.704g NaH
2PO
42H
2O, adding distil water be to 100mL, pH7.4), and its OD
350nmValue is about 1.0 (if 40mg micrococcus lyophilized powder is suspended in the phosphoric acid buffer of 50mL again its OD
350Value is about 1.6), subsequent use.
2. substrate is hatched: get the some milligrams of the pure article of T4 N,O-Diacetylmuramidase recombinant protein of above-mentioned preparation, respectively get 300uL after being diluted to different working concentration respectively, with the above-mentioned 2.7mL micrococcus substrate for preparing, negative control is a zero(ppm) water respectively.After placing 37 ℃ of incubators to hatch 1h each test tube, under the 350nm wavelength, measure the light absorption value (adding 300 μ L zero(ppm) water with the phosphate buffered saline buffer of 2.7ml 0.2M returns to zero) of solution, the mensuration result sees table 3:
The active detection of table 3 T4 N,O-Diacetylmuramidase recombinant protein bacteriolyze
| T4 N,O-Diacetylmuramidase working concentration (mg/mL) | OD 350The nm light absorption value | Contrast CK- |
| 0 | 1.721 | 1.693 |
| 0.0005 | 1.645 | 1.715 |
| 0.0010 | 1.554 | 1.673 |
| 0.0015 | 1.515 | 1.681 |
| 0.0020 | 1.443 | 1.696 |
| 0.0025 | 1.402 | 1.666 |
| 0.0030 | 1.364 | 1.697 |
| 0.0035 | 1.269 | 1.672 |
| 0.0040 | 1.242 | 1.673 |
Annotate: numeral is the MV of three test-results in the table
The present invention is first according to the mould codon of having a preference for of Rui Shi wood, synthetic a brand-new T4 lysozyme gene, and merge with the encoding sequence of Rui Shi mould cellobiohydrolase 1 secreting signal peptide of wood (17aa); The promotor of mould cellobiohydrolase 1 gene of Rui Shi wood and the terminator sequence of this gene have been cloned; And utilize above-mentioned various elements, on the basis of plasmid pPIC9K, rebuild brand-new, a mould efficient expression vector of Rui Shi wood induction type and secretor type; After in the T4 lysozyme gene being imported the mould cell of Rui Shi wood; Through methods such as resistance screening, active detections; Obtained to stablize and to efficiently express the mould engineering strain of reorganization Rui Shi wood of T4 N,O-Diacetylmuramidase; Through sequence of operations such as high density fermentation, recombinant protein purifications, the pure article tool of the reorganization T4 antalzyme protein biological activity of final preparation can be widely used in fields such as medical treatment, food, feed and scientific research again.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from know-why of the present invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Reference
1、Arisaka?F.，et?al.，The?international?journal?of?biochemistry?&?cellbiology，2003，35(1)：16-21
2、Cellitti?J.，et?al.，Protein?Sci.，2007，16(5)：842-51
3, Jiang Tianyi and Zhu Ping, biotechnology communication, 2007,18 (6): 1050-1052
4、Li?Tao，et?al.，Appl?Microbiol?Biotechnol.，2008，80：573-578
5、Bruno?C.，et?al.，Biotechnol.Lett.，2007，29：1195-1201
6、Hanna?K.，et?al.Biotechnology?and?Bioengineering，2006，94：407-415
7, Xu Jun etc., Acta Biochimica et Biophysica Sinica 2003,35 (5): 454-458
8、Ritva?S.，et?al.，Applied?and?Environmental?Microbiology，1997，63(12)：4938-4940
Claims (6)
1. one kind through the mould method that efficiently expresses and produce the T4 N,O-Diacetylmuramidase with the induction type mode of reorganization Rui Shi wood; Comprise the synthetic of T4 N,O-Diacetylmuramidase foreign gene, the structure of efficient expression vector, the screening of the mould recon of high expression level wood, the fermentation of the mould engineering bacteria of reorganization Rui Shi wood and the abduction delivering and the purifying of external source recombinant protein; It is characterized in that said T4 N,O-Diacetylmuramidase foreign gene is the gene codon optimized mistake, coding T4 antalzyme protein according to the mould preference of Rui Shi wood, described T4 antalzyme protein primary structure is the aminoacid sequence shown in the SEQ ID NO.1; Adopt Rui Shi mould cellobiohydrolase 1 gene promoter of wood and terminator sequence the T4 lysozyme gene to be incorporated in the mould genome of Rui Shi wood when making up efficient expression vector as homologous sequence; Added the encoding sequence of mould cellobiohydrolase 1 secreting signal peptide of Rui Shi wood before the 5 ' end of said T4 N,O-Diacetylmuramidase foreign gene.
2. the method for claim 1 is characterized in that, when making up efficient expression vector, the initial vector of employing is pPIC9K.
3. the expression plasmid carrier of the T4 antalzyme protein of recombinating, it is characterized in that its form and building process following:
(1) according to the dna nucleotide sequence and the amino acid sequence coded thereof of the T4 lysozyme gene shown in the GenBank:NC000866; Under the prerequisite that does not change its aminoacid sequence; According to the artificial dna encoding sequence that designs and synthesized new T4 N,O-Diacetylmuramidase maturation protein of the mould codon of having a preference for of Rui Shi wood, its sequence is shown in the SEQ ID NO.2;
(2) being primer with Primer1 and Primer2, is template with Rui Shi trichoderma strain QM9414 genomic dna, and pcr amplification obtains mould cellobiohydrolase 1 gene promoter of Rui Shi wood-secreting signal peptide nucleotide coding sequence, and wherein the Primer1 sequence is ac
AgatctAattctggagacggcttgttgaatc, the Primer2 sequence does
CtcgaagatgttcaTagcacgagctgtggccaagaaggc;
(3) being primer with Primer3 and Primer4, is template with Rui Shi trichoderma strain QM9414 genomic dna, and pcr amplification obtains the mould cellobiohydrolase 1 gene terminator sequence of Rui Shi wood, and wherein the Primer3 sequence does
TacaagaacttgtaaAggtcaccttctccaacatcaagttcg, the Primer4 sequence is ac
GtcgacCacgaagagcggcgattctacggg;
(4) with mould cellobiohydrolase 1 gene promoter of above-mentioned T4 lysozyme gene, Rui Shi wood-secreting signal peptide nucleotide coding sequence, the mould cellobiohydrolase 1 gene terminator sequence of Rui Shi wood mixed according to 1: 1: 1; Use Primer1 and Primer4 to carry out pcr amplification as primer; The PCR product of total length directly is inserted in the T site in the pEASY-T1 plasmid; Obtain containing the bacterial clone of the middle plasmid vector T4-T that exogenous dna fragment inserts; Then, through the nucleotide sequencing analysis, confirm that the dna sequence dna in the mould gene reading frame of T4 N,O-Diacetylmuramidase Rui Shi wood is correct and complete;
(5) carry out double digestion with restriction enzyme Bgl II and Sal I, the mould gene reading frame of the wood of the T4 N,O-Diacetylmuramidase Rui Shi in the plasmid vector T4-T in the middle of being connected is scaled off, utilize agarose gel electrophoresis to separate the purpose fragment; Handle plasmid pPIC9K with identical restriction enzyme, utilize agarose gel electrophoresis to separate the purpose fragment; Just obtain the mould expression vector T4-CBH9K of Rui Shi wood with linking together after the dna fragmentation mixing of above-mentioned two recovery and with ligase enzyme, be the expression plasmid carrier of reorganization T4 antalzyme protein.
4. the engineering strain of an express recombinant T4 antalzyme protein is characterized in that, comprises the described expression plasmid carrier of claim 3.
5. engineering strain according to claim 4 is characterized in that, host cell is Rui Shi trichoderma strain QM9414.
6. adopt the method for claim 4 or 5 described engineering strain fermentative prodn recombinant proteins, comprising:
(1) seed liquor preparation: at first with mycelium inoculation on the PDA substratum; Cultivated 6～7 days for 27～28 ℃, again spore is dissolved in the sterilized water, be inoculated in the liquid MM substratum with 1～2% volume ratio; 27～28 ℃ of shaking culture 72～96 hours are as the seed liquor of high density fermentation;
(2) yeast culture: in the wooden mould seed liquor of 8～10% ratio inoculation step (1), 27～28 ℃ of aeration-agitations were cultivated 72～96 hours in the MM substratum, and pH maintains 5.5～6;
(3) protein induced expression: yeast culture is after 72～96 hours, and stream adds the Mierocrystalline cellulose that is dissolved in 50wt% in the MM substratum, makes the substratum cellulose concentration maintain 2～3wt%; Dissolved oxygen amount is greater than 20%; Temperature maintenance is at 27～28 ℃, and pH maintains 5.5～6, continues the vibration inducing culture 3～4 days;
The MM culture medium prescription of in said process, using is: every liter contains: glucose 20g, (NH
4)
2SO
45g, KH
2PO
415g, MgSO
47H
2O 0.6g, CaCl
20.6g, FeSO
47H
2O 0.005g, MnSO
4H
2O 0.0016 g, ZnSO
47H
2O 0.0014Gg, CoCl
20.002g, pH5.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101584539A CN101831452B (en) | 2010-04-22 | 2010-04-22 | Method for efficiently expressing and producing T4 lysozyme through recombinant trichoderma reesei in inductive mode |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101584539A CN101831452B (en) | 2010-04-22 | 2010-04-22 | Method for efficiently expressing and producing T4 lysozyme through recombinant trichoderma reesei in inductive mode |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101831452A CN101831452A (en) | 2010-09-15 |
| CN101831452B true CN101831452B (en) | 2012-09-05 |
Family
ID=42715671
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010101584539A Expired - Fee Related CN101831452B (en) | 2010-04-22 | 2010-04-22 | Method for efficiently expressing and producing T4 lysozyme through recombinant trichoderma reesei in inductive mode |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101831452B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3523415A1 (en) * | 2016-10-04 | 2019-08-14 | Danisco US Inc. | Protein production in filamentous fungal cells in the absence of inducing substrates |
| CN107794274A (en) * | 2017-10-27 | 2018-03-13 | 杭州欧亘生物科技有限公司 | A kind of people source antalzyme protein production technology |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101173260A (en) * | 2006-10-31 | 2008-05-07 | 刘德虎 | Representation of high disinfection vitality T4 lysozyme in yeast and producing method thereof |
-
2010
- 2010-04-22 CN CN2010101584539A patent/CN101831452B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101173260A (en) * | 2006-10-31 | 2008-05-07 | 刘德虎 | Representation of high disinfection vitality T4 lysozyme in yeast and producing method thereof |
Non-Patent Citations (3)
| Title |
|---|
| Klaus DÜring等.The non-enzymatic microbicidal activity of lysozymes.《FEBS Letters》.1999,第449卷(第2-3期),93^100. * |
| Wen-Jing Hao,Gang-Qiang Li等.Induction and expression of T4 lysozyme gene in Pichia pastoris.《Journal of Chinese Pharmaceutical Sciences》.2007,第16卷33-37. * |
| 王艳君,杨谦.一种快速获得基因密码子偏爱性改造的方法——SOE-PCR.《中国生物工程杂志》.2008,第28卷(第8期),96-99. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101831452A (en) | 2010-09-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102517304B (en) | Optimized gene of recombinant glucose oxidase and expression vector and application of optimized gene | |
| CN103045636B (en) | Method for efficiently expressing antibacterial peptide NZ2114 in recombinant pichia pastoris | |
| CN103937830B (en) | A kind of recombinant bacterium of efficient secretory expression Nattokinase | |
| CN104524564B (en) | A kind of crucian Simplex Virus combination vaccine preparation and preparation method and application | |
| CN101070530B (en) | Low-temperature alkaline phosphatidase A1 and coding gene thereof | |
| KR102186997B1 (en) | Novel method of protein purification | |
| CN105018448A (en) | Heat-resisting acidic cellulase of fungus source and gene and application thereof | |
| CN108102936B (en) | Kluyveromyces lactis mutant strain, glycosidase thereof and application thereof | |
| CN100348720C (en) | Mannase and its coding gene and uses | |
| CN101845454A (en) | Method for expressing pseudoplectania nigrella mature peptide in recombinant pichia pastoris | |
| CN104278017A (en) | Recombinant expression method of human lysozyme | |
| CN101831452B (en) | Method for efficiently expressing and producing T4 lysozyme through recombinant trichoderma reesei in inductive mode | |
| CN101831451B (en) | Method for efficiently expressing and producing T4 lysozyme through recombinant hansenula polymorpha in constitutive mode | |
| CN103305426B (en) | The mutant strain of cellulase-producing, the mutant strain of high efficiency expressing destination protein and construction process thereof and application | |
| CN107794274A (en) | A kind of people source antalzyme protein production technology | |
| CN108192903A (en) | A kind of alkalescent xylanase and its encoding gene and application | |
| CN101942406A (en) | Marine nocardiopsissp.HY-G and beta-glucosidase produced by same | |
| CN104131021A (en) | Antibacterial peptide coexpression vector, construction and expression method thereof | |
| CN101182475B (en) | Pichia yeast highly-effective expression of elastase as well as construction method and uses thereof | |
| CN104480027B (en) | The Aspergillus niger strain of a kind of high proteinase yield K and application thereof | |
| CN104560833B (en) | A kind of basophilic micrococcus luteus and its alkalescent xylanase and the application of generation | |
| CN102533833A (en) | Construction method for streptomycete expression plasmids and production method for keratinase | |
| CN106995793A (en) | A kind of fermentation process in high density for recombinating bG-CSF engineering bacteria | |
| CN110423711B (en) | Low-temperature chitinase-producing strain from Antarctic and fermentation method thereof | |
| CN110527690A (en) | A kind of heat resistant type tannase and its application |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120905 |