CN102559672B - Recombinant sea cucumber lysozyme N-terminal peptide, preparation method and application thereof - Google Patents
Recombinant sea cucumber lysozyme N-terminal peptide, preparation method and application thereof Download PDFInfo
- Publication number
- CN102559672B CN102559672B CN201210055509.7A CN201210055509A CN102559672B CN 102559672 B CN102559672 B CN 102559672B CN 201210055509 A CN201210055509 A CN 201210055509A CN 102559672 B CN102559672 B CN 102559672B
- Authority
- CN
- China
- Prior art keywords
- sea cucumber
- antalzyme
- recombinant
- polypeptide
- lysozyme
- 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
- 241000251511 Holothuroidea Species 0.000 title claims abstract description 79
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 63
- 238000002360 preparation method Methods 0.000 title claims description 16
- 108010014251 Muramidase Proteins 0.000 title abstract description 13
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 title abstract description 13
- 102000016943 Muramidase Human genes 0.000 title abstract description 10
- 229960000274 lysozyme Drugs 0.000 title abstract description 10
- 235000010335 lysozyme Nutrition 0.000 title abstract description 10
- 239000004325 lysozyme Substances 0.000 title abstract description 10
- 229920001184 polypeptide Polymers 0.000 claims abstract description 58
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 58
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 40
- 230000014509 gene expression Effects 0.000 claims abstract description 19
- 238000005457 optimization Methods 0.000 claims abstract description 8
- 238000003259 recombinant expression Methods 0.000 claims abstract description 8
- 239000013613 expression plasmid Substances 0.000 claims abstract description 6
- 241000894006 Bacteria Species 0.000 claims description 50
- 239000007788 liquid Substances 0.000 claims description 27
- 108010064696 N,O-diacetylmuramidase Proteins 0.000 claims description 24
- 239000000047 product Substances 0.000 claims description 21
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 17
- 238000010353 genetic engineering Methods 0.000 claims description 16
- 239000012634 fragment Substances 0.000 claims description 14
- 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 claims description 12
- 235000015097 nutrients Nutrition 0.000 claims description 12
- 239000013612 plasmid Substances 0.000 claims description 11
- 238000004519 manufacturing process Methods 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 230000003115 biocidal effect Effects 0.000 claims description 8
- 238000000746 purification Methods 0.000 claims description 8
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 7
- 238000005215 recombination Methods 0.000 claims description 7
- 230000006798 recombination Effects 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
- 239000000654 additive Substances 0.000 claims description 6
- 238000010612 desalination reaction Methods 0.000 claims description 6
- 230000004927 fusion Effects 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 239000002953 phosphate buffered saline Substances 0.000 claims description 6
- 238000000108 ultra-filtration Methods 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 5
- 230000001939 inductive effect Effects 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 229910001453 nickel ion Inorganic materials 0.000 claims description 5
- 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 claims description 4
- 239000005452 food preservative Substances 0.000 claims description 4
- 235000019249 food preservative Nutrition 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 229920005654 Sephadex Polymers 0.000 claims description 3
- 239000012507 Sephadex™ Substances 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 3
- 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 claims description 3
- 238000009313 farming Methods 0.000 claims description 3
- 238000005227 gel permeation chromatography Methods 0.000 claims description 3
- 229940049547 paraxin Drugs 0.000 claims description 3
- 239000013504 Triton X-100 Substances 0.000 claims description 2
- 229920004890 Triton X-100 Polymers 0.000 claims description 2
- 238000000855 fermentation Methods 0.000 abstract description 23
- 230000004151 fermentation Effects 0.000 abstract description 23
- 230000001580 bacterial effect Effects 0.000 abstract description 9
- 241000588724 Escherichia coli Species 0.000 abstract description 8
- 241000965254 Apostichopus japonicus Species 0.000 abstract description 7
- 230000000845 anti-microbial effect Effects 0.000 abstract description 7
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 3
- 230000001131 transforming effect Effects 0.000 abstract description 2
- 239000003674 animal food additive Substances 0.000 abstract 1
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 230000006698 induction Effects 0.000 description 18
- 239000000523 sample Substances 0.000 description 11
- 230000009182 swimming Effects 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 8
- 230000000968 intestinal effect Effects 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 7
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 7
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 6
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000012530 fluid Substances 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000013016 damping Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 241000607598 Vibrio Species 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 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 2
- 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 2
- 241000607528 Aeromonas hydrophila Species 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 241000191938 Micrococcus luteus Species 0.000 description 2
- 238000009004 PCR Kit Methods 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 206010047400 Vibrio infections Diseases 0.000 description 2
- 241000607272 Vibrio parahaemolyticus Species 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000012148 binding buffer Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 235000014103 egg white Nutrition 0.000 description 2
- 210000000969 egg white Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 150000002460 imidazoles Chemical class 0.000 description 2
- 239000008176 lyophilized powder Substances 0.000 description 2
- 238000012803 optimization experiment Methods 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000000452 restraining effect Effects 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 235000014102 seafood Nutrition 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 210000001082 somatic cell Anatomy 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- WFZFMHDDZRBTFH-CZEFNJPISA-N 2-[(e)-2-(5-carbamimidoyl-1-benzofuran-2-yl)ethenyl]-1-benzofuran-5-carboximidamide;dihydrochloride Chemical compound Cl.Cl.NC(=N)C1=CC=C2OC(/C=C/C=3OC4=CC=C(C=C4C=3)C(=N)N)=CC2=C1 WFZFMHDDZRBTFH-CZEFNJPISA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000143060 Americamysis bahia Species 0.000 description 1
- 241000258957 Asteroidea Species 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 101000874334 Dalbergia nigrescens Isoflavonoid 7-O-beta-apiosyl-glucoside beta-glycosidase Proteins 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 241000257465 Echinoidea Species 0.000 description 1
- 101000757733 Enterococcus faecalis (strain ATCC 700802 / V583) Autolysin Proteins 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- 101001091385 Homo sapiens Kallikrein-6 Proteins 0.000 description 1
- 102100034866 Kallikrein-6 Human genes 0.000 description 1
- 241000256766 Lophodermium medium Species 0.000 description 1
- 101000757734 Mycolicibacterium phlei 38 kDa autolysin 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-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
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000544286 Vibrio anguillarum Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000001857 anti-mycotic effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 239000002543 antimycotic Substances 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 229960001438 immunostimulant agent Drugs 0.000 description 1
- 239000003022 immunostimulating agent Substances 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000011177 media preparation Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000013595 supernatant sample Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 108010046845 tryptones Proteins 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to recombinant expression for an N-terminal peptide of a lysozyme (SjLys) gene (GenBank registration number: EF036468) which is sourced from a sea cucumber (Stichopus japonicus). The recombinant expression comprises the following steps: first, establishing a recombinant expression plasmid pET-32a-SjLys-N; and then, transforming the recombinant expression plasmid into an escherichia coli Rosetta (DE3) pLysS cell, and thus obtaining a gene engineering bacterial strain capable of efficiently expressing the recombinant sea cucumber lysozyme N-terminal peptide. Through optimization of a fermentation medium and fermentation conditions, an expression product of the lysozyme N-terminal peptide prepared from the gene engineering bacterial strain has more than 50 percent of solubility and has a high product yield; the prepared recombinant sea cucumber lysozyme N-terminal peptide has a broad-spectrum antimicrobial activity; and especially, after the product is heated for 40min at the temperature of 100 DEG C, the antibacterial activity thereof is unchanged, which is very beneficial to the development of sea cucumber lysozyme polypeptide as a feed additive.
Description
Technical field
The invention belongs to biological technical field, relate to the engineering bacteria preparation method of the high efficient expression of recombinant sea cucumber antalzyme N end polypeptide, optimization for fermentation technology and obtain the soluble polypeptide product of high yield, and the product of preparation is carried out to the research of broad spectrum antibiotic activity.
Background technology
N,O-Diacetylmuramidase is a kind of alkaline enzyme that can be hydrolyzed mucopolysaccharide, and the hydrolysis of the β-Isosorbide-5-Nitrae glycosidic link in its energy catalysis bacteria cell wall between mucopolysaccharide-acetylmuramic acid and N-Acetyl-D-glucosamine, causes lysis, and content is overflowed and made bacterium death.It is extensively present in tissue, body fluid and the secretory product of nature animals and plants and microorganism, in the immune defense system of living organism, plays a significant role.N,O-Diacetylmuramidase is widely used in the industry such as medicine, food and feeds at present as natural antiseptic-germicide, immunostimulant and sanitas.In recent years along with marine organisms fish, shrimps, shellfish and precious marine product, as the rapid growth of the cultured output such as sea cucumber, abalone, the consequent is that quality deterioration, the cultivation level during sea farming is produced is low, disease is spread unchecked and the problem such as environmental pollution.In aquaculture and Production of Livestock and Poultry, use at present problem that microbiotic brings also more and more to receive people's concern, the one, resistance problem, the 2nd, drug residue problem, its side effect is very important, and it is the raising that cost exchanges sea-food, animal products production performance for that people do not wish to sacrifice human health.Under this severe situation, the advantage of N,O-Diacetylmuramidase aspect animal cultivation and the promoter action aspect prophylactic treatment have shown its prominent position, and its green, safety, non-harmful feature have represented the direction of following fodder additives and animal health-care product.
Conventionally can be divided into 6 classes according to the difference of N,O-Diacetylmuramidase space structure, immunological characteristic and catalytic activity: c-type (chicken-type) N,O-Diacetylmuramidase, g-type (goose-type) N,O-Diacetylmuramidase, i-type (invertebrate-type) N,O-Diacetylmuramidase; Plant-sourced (plant lysozyme) N,O-Diacetylmuramidase; Microbial source (bacterial lysozyme) N,O-Diacetylmuramidase; Phage (phage lysozyme) N,O-Diacetylmuramidase.Along with people invest ocean the sight of developing new drug and functional foodstuff, the research interest of i-type N,O-Diacetylmuramidase is also grown to even greater heights in recent years, in succession in seashells, shrimp, starfish, sea urchin, sea cucumber, found i-type N,O-Diacetylmuramidase.
The N,O-Diacetylmuramidase that commercialization has been used at present mainly extracts from egg white, belongs to c-type N,O-Diacetylmuramidase.Because source is limited, complex manufacturing, N,O-Diacetylmuramidase output is limited, holds at high price, and is difficult to meet the increasing market requirement.Therefore, utilize the high efficient expression N,O-Diacetylmuramidase of genetically engineered recombinant technology to become very important means, especially produce sea cucumber i-type N,O-Diacetylmuramidase and there is very important using value.Research is found, sea cucumber i-type N,O-Diacetylmuramidase is different from egg white c-type N,O-Diacetylmuramidase, the latter only plays anti-microbial effect to gram-positive microorganism, and sea cucumber i-type N,O-Diacetylmuramidase is not only to gram-positive microorganism, and Gram-negative bacteria is all had to significant anti-microbial effect, especially the pathogenic bacteria (vibrios and pseudomonas) that conventionally causes aquatic animal serious plant disease is had to obvious antibacterial effect.Meanwhile, sea cucumber i-type N,O-Diacetylmuramidase have stronger antimycotic, antiviral, increase the abilities such as immunizing power, and there is the features such as good biocompatibility,, nontoxicity non-stimulated to organizing.
Sea cucumber i-type N,O-Diacetylmuramidase (hereinafter to be referred as sea cucumber antalzyme) gene be isolated and identified first in our laboratory in 2007 (referring to Yang Xijian, etc. the constructional feature of sea cucumber i-type lysozyme gene and coded product thereof. Chinese biological chemistry and molecular biosciences journal; 2007,23(7): 542-547).In recent years; we carry out respectively recombinant expressed by sea cucumber antalzyme gene again in intestinal bacteria and Pichia pastoris; and the anti-microbial activity of recombinant protein is studied (referring to Wang Xiuxia; Deng. the recombinant expressed and antimicrobial spectrum analysis of extra large stichopus japonicus i-type lysozyme gene. biotechnology journal; 2009,25 (2): 189-194; Gu Yuefeng, etc. sea cucumber antalzyme gene clone and the expression and purification in pichia spp. Dalian Polytechnic University's journal, 2010,29 (5): 317-320).But the problem existing at present, the one, recombinant sea cucumber antalzyme gene is expressed in Bacillus coli cells, there is (being that zymoprotein does not dissolve) with inclusion body form in its antalzyme protein, the follow-up denature and renature complex operation to this albumen, and also the production of enzyme obtaining is low, active unstable; The 2nd, recombinant sea cucumber antalzyme gene expressing quantity in Pichia pastoris is very low.These two bottleneck problems have seriously hindered the process of sea cucumber antalzyme research to industrialization test, and domestic and international several laboratory all runs into Similar Problems in the time that study of various lysozyme gene is expressed.Through analysis to sea cucumber antalzyme gene structure, the N end regions of the finding sea cucumber antalzyme beta-glycosidase activity of may having encoded, and the C end regions isopeptide enzymic activity of may having encoded.Therefore, we are divided into two sections by sea cucumber antalzyme gene at design, carry out respectively being recombinant expressedly prepared into sea cucumber antalzyme N end and C holds polypeptide, then verify its characteristic and function, and compare and analyze with sea cucumber antalzyme.The invention relates to preparation method and the application of recombinant sea cucumber antalzyme N end polypeptide.
Summary of the invention
The object of the present invention is to provide a kind of gene engineering preparation method with broad spectrum antibiotic activity, high efficient expression sea cucumber antalzyme polypeptide.The technical solution used in the present invention is: N,O-Diacetylmuramidase (SjLys) gene (GenBank number of registration: EF036468) to the extra large stichopus japonicus (Stichopus japonicus) that derives from autonomous isolation identification carries out the recombinant expressed of N end polypeptide.Utilize the synthetic primer of design, the gene of amplification sea cucumber antalzyme polypeptide; By the prokaryotic expression carrier pET-32a-SjLys-N building, transform Rosetta (DE3) pLysS cell, obtain the genetic engineering bacterium of a plant height efficient expression recombinant sea cucumber antalzyme N end polypeptide.The lysozyme polypeptide of producing with described genetic engineering bacterium, through the optimization of fermention medium and fermentation condition, its expression product has more than 50% solubility, and product yield is high.The recombinant sea cucumber antalzyme N end polypeptide of producing has broad spectrum antibiotic activity, and especially, through 100 ℃ of heating 40min, the anti-microbial activity of this polypeptide products remains unchanged substantially.Studies have shown that, anti-microbial activity, stability and the production efficiency of sea cucumber antalzyme N end polypeptide are all better than sea cucumber antalzyme, can prepare fodder additives, food preservatives, antibiotic alternative medicine etc. without endangering to be applied to.
An aspect of of the present present invention is to protect a kind of primer sequence of recombinant sea cucumber antalzyme N end polypeptide, and its primer sequence is respectively, forward primer: SEQ ID NO:1; Reverse primer: SEQ ID NO:2.
Another aspect of the present invention is to protect a kind of plasmid of recombinant sea cucumber antalzyme N end polypeptide; its preparation method is as follows: take sea cucumber antalzyme SjLys gene as template; forward primer by above-mentioned: SEQ ID NO:1 and reverse primer: the gene fragment that SEQ ID NO:2 increases is connected in coli expression carrier pET32a (+), builds recombinant expression plasmid.
Another aspect of the present invention is to protect a kind of genetic engineering bacterium of expressing recombinant sea cucumber antalzyme N end polypeptide, and its preparation method comprises: above-mentioned plasmid is transformed to Rosetta (DE3) pLysS host cell and obtain recombination engineering bacteria.
Another aspect of the present invention is to protect a kind of preparation method of recombinant sea cucumber antalzyme N end polypeptide, and it comprises:
1. abduction delivering: cultivate above-mentioned recombination engineering bacteria, and use IPTG abduction delivering;
2. separate and purifying: collect 1. products obtained therefrom of step, through nickel ion affinity chromatograph column purification, and concentrated with ultra-filtration membrane; To after concentrating sample desalination, make finished product again.
Concrete, in above-mentioned method, the condition of its abduction delivering and separation and purification comprises:
3. abduction delivering
By recombination engineering bacteria mentioned above, in the liquid nutrient medium of optimizing, 37 ℃, 160rpm shaking culture are to OD
600reach 0.6-0.8, then be 0.5mmol/L to adding IPTG in culture system to final concentration, 28 ℃, 150rpm inducing culture 8h, collect fermented liquid;
Wherein, the liquid nutrient medium of optimization refers to: in LB substratum, add two kinds of microbiotic, i.e. and 100mg/L penbritin and 34mg/L paraxin, and add 5g/L glucose, 0.3g/L MgSO
47H
2o and 1g/L K
2hPO
43H
2o;
4. separate and purifying
The fermented liquid that 3. step is collected is centrifugal, collect and wash thalline, multigelation, with the resuspended thalline of phosphate buffered saline buffer, and after adding Triton X-100 to be 1% to final concentration, through the broken bacterium of ice-bath ultrasonic, centrifugal collection supernatant liquor, through nickel ion affinity chromatograph post His Trap HP column purifying, collect the fusion rotein component of recombinant sea cucumber antalzyme polypeptide, albumen is concentrated into 8～10mg/mL by the Millipore ultra-filtration membrane that is 5kDa with molecular weight cut-off; Again by concentrating sample through the desalination of Sephadex G-25 gel chromatography, collect fusion rotein component, lyophilize, obtains the product of recombinant sea cucumber antalzyme N end polypeptide.
Another aspect of the present invention is to protect a kind of recombinant sea cucumber antalzyme N end polypeptide that utilizes above-mentioned method to obtain.
Another aspect of the present invention is to protect a kind of fodder additives, food preservatives, antibiotic alternative medicine and application in sea farming production field that utilizes above-mentioned recombinant sea cucumber antalzyme N end polypeptide preparation.
Following each experimental implementation that the invention described above relates to, comprise: the Examination on experimental operations such as gene fragment is connected in coli expression carrier pET32a (+), plasmid transforms Rosetta (DE3) pLysS host cell, nickel ion affinity chromatograph column purification, ultra-filtration membrane are concentrated, sample desalination are the conventional experimental technique of those skilled in the art, therefore again do not repeat part described in specific embodiment in concrete visible the present invention.
Character of innovation of the present invention is:
1. high efficient expression and preparation solubility recombinant sea cucumber antalzyme N end polypeptide, has solved the key issues such as the albumen solubility that current sea cucumber antalzyme industrialization runs into is low, enzymic activity is low.
2. genetic engineering bacterium Restruction sea cucumber antalzyme N end polypeptide provided by the invention, has output high, and working condition and step are simple, and reaction conditions is easy to control, and the advantage that production cost is low, is therefore applicable to large-scale production.
3. recombinant sea cucumber antalzyme N end polypeptide has broad spectrum antibiotic activity, and has good thermostability and enzyme stability alive, and safe and harmless, can be applicable to prepare fodder additives, food preservatives, antibiotic alternative medicine etc.
Accompanying drawing explanation
Fig. 1 is the gene fragment of pcr amplification SjLys-N; Wherein swimming lane, M:50bp DNA ladder marker; 1:PCR amplified production;
Fig. 2 is the Western blotting analytical results figure of recombinant sea cucumber antalzyme N end polypeptide; Wherein swimming lane, M: precisely protein molecular weight standard; The thalline of bacterial strain fermentation liquor before 1:IPTG induction; The thalline of bacterial strain fermentation liquor after 2:IPTG induction; 3:Western blotting detects the sample in swimming lane 1; 4:Western blotting detects the sample in swimming lane 2;
Fig. 3 is the recombinant sea cucumber antalzyme N end abduction delivering of polypeptide and the SDS-PAGE result figure of purifying; Wherein swimming lane, M: protein molecular weight standard; The thalline of bacterial strain fermentation liquor before 1:IPTG induction; The thalline of bacterial strain fermentation liquor after 2:IPTG induction; 3: the throw out after the bacterial strain fermentation liquor somatic cells of induction is broken; 4: the supernatant liquor after the bacterial strain fermentation liquor somatic cells of induction is broken; 5: the sample in swimming lane 4 is through Ni
2+the recombinant protein of-NTA affinity chromatography column purification.
Embodiment
Following non-limiting example can make the present invention of those of ordinary skill in the art's comprehend, but does not limit the present invention in any way.
Extra large stichopus japonicus (Stichopus japonicus) used in following embodiment, comes from area, Changhai County, Dalian, and the GenBank number of registration of its sea cucumber antalzyme (SjLys) gene is EF036468;
Primer sequence is synthetic, DNA sequencing is to be completed by Huada Gene Research Center, Beijing;
It is to be completed by TaKaRa Biotechnology (Dalian) company that Western blotting analyzes;
Intestinal bacteria Rosetta (DE3) pLysS competent cell and expression vector pET-32a (+) are purchased from Novagen (U.S.) company;
Bacillus coli DH 5 alpha, cloning vector pMD18-T, RNAiso Plus reagent, TaKaRa One Step RNA PCR Kit (AMV) test kit, restriction endonuclease, T
4dNA ligase, Taq archaeal dna polymerase, DNA ladder marker and albumen lower molecular weight marker are all purchased from TaKaRa Biotechnology (Dalian) company;
Plasmid extraction and agarose gel electrophoresis DNA reclaim test kit all purchased from TIANGEN Biotech (Beijing) Co., Ltd.;
HisTrap HP column is purchased from GE Healthcare (U.S.) company;
Other reagent are domestic analytical pure.
The preparation of the genetic engineering bacterium of the high efficient expression recombinant sea cucumber antalzyme of embodiment 1 N end polypeptide
1. the amplification of sea cucumber antalzyme N end polypeptide gene sequence
(1) according to the gene order of sea cucumber antalzyme, design two pairs of Auele Specific Primers, in forward and reverse sequence, introduce respectively Nco I and EcoR I restriction enzyme site.Its sequence is:
Forward primer: SEQ ID NO:1
TGA
CCATGGCTATGCAAGTTCCTTCTGAT
Reverse primer: SEQ ID NO:2
CCAG
GAATTCTCACTTCAGCCTAGCATCC
Pcr amplified fragment scope is from sea cucumber antalzyme amino acid Gln21 to Lys69, and total length is that 147bp(contains 49aa), be the gene fragment of SjLys-N.
(2) get fresh extra large stichopus japonicus intestines sample, according to RNAiso Plus reagent explanation extracted total RNA.
(3) take the total RNA of this Intestinum Stichopi japonici as template, use TaKaRa One Step RNA PCR Kit (AMV) to carry out RT-PCR gene amplification, its reaction system is:
RT-PCR loop parameter is: 50 ℃ of 30min, 94 ℃ of 2min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 45s, 35 circulations; 72 ℃ are extended 10min.
After question response finishes, get 5 μ L PCR products, with 1.7% agarose gel electrophoresis testing goal gene, and object band is reclaimed to test kit by agarose gel electrophoresis DNA and reclaim purifying.Electrophoretic analysis result shows (Fig. 1), and amplification gene fragment SjLys-N presents specificity bright band near 150bp, basically identical with expection fragment (147bp) size.
2. build high efficient expression recombinant sea cucumber antalzyme N end polypeptide gene engineering bacteria
(1) SjLys-N gene fragment amplification being obtained, after Nco I and EcoR I restriction enzymes double zyme cutting, purifying reclaims its goal gene fragment; Again with linearizing pMD18-T carrier after Nco I and EcoR I double digestion by T
4dNA ligase connects, 16 ℃ of reaction overnight, construction recombination plasmid pMD18-SjLys-N.Get connection product 5 μ L and add in the competent cell of the 50 μ L bacillus coli DH 5 alphas that thaw, ice bath 30min; Put into again 42 ℃ of heating 45s, put immediately 2min in ice; Then add SOC liquid nutrient medium 945 μ L, shaking culture 60min at 37 ℃, obtains transforming the e.colidh5αcell conversion fluid of sea cucumber antalzyme N end polypeptide gene.
(2) appropriate this conversion fluid is coated containing 37 ℃ of incubated overnight in the LB solid medium of 100mg/L penbritin (Amp), picking list bacterium colony extracts recombinant plasmid after the LB liquid nutrient medium containing 100mg/L Amp is cultivated respectively.Carry out enzyme with Nco I and EcoR I Restriction Enzyme and cut rear electrophoresis, obtain the gene fragment of SjLys-N.
LB liquid nutrient medium preparation: Tryptones 10g, yeast extract 5g, NaCl10g; Each components dissolved, in 900mL deionized water, is adjusted to pH to 7.0 with 1mol/L NaOH, then moisturizing is to 1000mL, high pressure steam sterilization.In addition, LB solid medium need add agar powder 15g/L.Medium sterilization is cooled to 60 ℃ of left and right, adds corresponding microbiotic by concentration requirement.
(3) SjLys-N gene fragment is connected with coli expression carrier pET32a (+), builds recombinant expression plasmid pET32a-SjLys-N, be transformed into bacillus coli DH 5 alpha, make the genetic engineering bacterium containing sea cucumber antalzyme N end polypeptide.This genetic engineering bacterium is entrusted to professional biotech company's order-checking, confirm that they contain complete SjLys-N gene fragment.
(4) the genetic engineering bacterium extracting recombinant plasmid pET32a-SjLys-N having built from step 2 (3), be transformed in intestinal bacteria Rosetta (DE3) pLysS competent cell, and get 50 μ L and coat on the LB solid medium that contains 100mg/L penbritin (Amp) and 34mg/L paraxin (Cam), 37 ℃ of incubated overnight.Respectively picking list colony inoculation is in containing 100mg/LAmp, and in the LB liquid nutrient medium of 34mg/L Cam, 37 ℃ of shaking culture 14～16h, extract recombinant plasmid and do PCR and enzyme and cut to detect and identify that whether the gene fragment size of inserting is correct.Picking transformant carries out sequencing again, the exactness of checking recombinant expression plasmid reading frame, and the sea cucumber antalzyme N that obtains high efficient expression holds the genetic engineering bacterium of polypeptide, called after pET32a-SjLys-N/Rosetta (DE3) pLysS.
Embodiment 2 abduction delivering and the Western blottings of recombinant sea cucumber antalzyme N end polypeptide in intestinal bacteria detect
The genetic engineering bacterium that builds correct sea cucumber antalzyme N end polypeptide is inoculated in to the Amp containing 100mg/L, in the LB liquid nutrient medium of 34mg/L Cam and 10g/L glucose, 160rpm shaking culture 14～16h at 37 ℃.Then be inoculated in the Amp containing 100mg/L according to the inoculum size of volume ratio 1%, 34mg/L Cam, and in the LB liquid nutrient medium of 5g/L glucose, 160rpm shaking culture is to OD at 37 ℃
600reach 0.6～0.7, take out 1mL bacterium liquid as the front control sample of induction.Adding inductor IPTG is 0.5mmol/L to fermented liquid final concentration again, 120rpm inducing culture 10h at 28 ℃, take out 1mL bacterium liquid as induction after sample.The thalline of last centrifugal fermented liquid collecting precipitation, by the expression situation of this recombinant protein of 15%SDS-PAGE Preliminary detection.Electrophoresis result shows (swimming lane 1 and 2 in Fig. 2), and restructuring SjLys-N obviously has more 1 specificity bright band in about 23kDa left and right.
In order to verify whether recombinant plasmid pET32a-SjLys-N is really expressed in intestinal bacteria, utilize Western blotting analyze in Penta-His antibody capable and the feature of recombinant sea cucumber polypeptide generation specific binding detect.The thalline sample of the sample before the induction of above-mentioned restructuring SjLys-N genetic engineering bacterium and induction secondary fermentation liquid is carried out, after 15%SDS-PAGE electrophoresis, being transferred to PVDF(poly(vinylidene fluoride)) on film, be placed on containing in 1.5%BSA sealing damping fluid, 4 ℃ keep flat and spend the night.The Penta-His antibody that adds 1:1000 dilution, carries out antibody response 1h one time.With containing 0.1%(v/v) Tris-HCl damping fluid (pH7.4) washing of Tween20 2 times, rinse after 3 times with washing Tris-HCl damping fluid (pH7.4) again, add the rabbit anti-mouse igg monoclonal antibody of the HRP mark of the dilution of 1:1000, carry out secondary antibodies reaction 1h.Repeat again to wash film once after, finally add TrueBlue peroxidase substrate, colour developing 1min post analysis.Analytical results explanation (swimming lane 3 and 4 in Fig. 2), can there is specific combination with Penta-His antibody in 23kDa left and right in restructuring SjLys-N albumen, thereby prove that this recombinant protein has really obtained correction in intestinal bacteria.
Fermentation optimization and the separation and purification of the genetic engineering bacterium of embodiment 3 recombinant sea cucumber antalzyme N end polypeptide
1. the optimization of fermention medium
The basic medium of the genetic engineering bacterium of fermentation culture recombinant sea cucumber antalzyme N end polypeptide is the LB liquid nutrient medium that contains microbiotic 100mg/LAmp and 34mg/L Cam.On this basis, carry out medium optimization experiment, comprised and add glucose (2g/L, 5g/L, 10g/L; ), MgSO
47H
2o (0.1g/L, 0.3g/L, 0.5g/L; ), and K
2hPO
43H
2o(0.5g/L, 1g/L, 1.5g/L; ).Add IPTG inductor to carry out the abduction delivering of recombinant polypeptide albumen, the fermented liquid obtaining, after cytoclasis, is collected supernatant liquor and is analyzed the wherein content of soluble proteins.Analyze through SDS-PAGE, determine that Optimal compositions of fermentation medium formula is: containing 100mg/L Amp, in the LB substratum of 34mg/L Cam, add 5g/L glucose, 0.3g/L MgSO
47H
2o, 1g/L K
2hPO
43H
2o.
2. the optimization of fermentation inducement condition
The optimization experiment of optimal conditions of fermentation comprises: and induction leavening temperature (25 ℃, 28 ℃, 30 ℃, 35 ℃), IPTG inductor concentration (0.2mmol/L, 0.5mmol/L, 0.8mmol/L, 1.0mmol/L), induction fermentation time (4h, 6h, 8h, 10h), shaking table revolution (220rpm after induction, 180rpm, 150rpm, 120rpm).The genetic engineering bacterium of sea cucumber antalzyme N end polypeptide is under the condition of above-mentioned optimal medium formula, and 37 ℃, 160rpm shaking culture are to OD
600reach 0.6-0.8, add inductor induction fermentation, test each inductive condition.The fermented liquid finally obtaining, after cytoclasis, is collected supernatant liquor and is analyzed the wherein content of soluble proteins.Analyze through SDS-PAGE, determine that best induction fermentation condition is: IPTG concentration is 0.5mmol/L, 28 ℃, 150rpm, inducing culture 8h.
3. under top condition, induce fermentation results
Under above-mentioned optimal medium formula and best induction fermentation condition, detect the whether efficiently abduction delivering of genetic engineering bacterium of recombinant sea cucumber antalzyme N end polypeptide, and whether recombinant protein after expression has solubility.Adopt biological software Bandscan5.0 to analyze the electrophoresis result of SDS-PAGE, the thalline expression amount after restructuring SjLys-N induction accounts for 75% left and right (swimming lane 2 in Fig. 3) of total protein content; And this thalline contains the amount of restructuring SjLys-N albumen in its supernatant liquor of cytoclasis post analysis and throw out, result is containing target protein more than 50% (swimming lane 4 in Fig. 3) in its supernatant liquor.This SjLys-N polypeptide that shows to recombinate can high efficient expression in intestinal bacteria, and most of albumen of expressing can exist with soluble form.
4. the separation of product and purifying
Low-temperature centrifugation is collected the thalline (4 ℃, 1000rpm, 10min) after induction fermentation, with phosphate buffered saline buffer (1 × PBS, pH7.4) washing thalline twice, by thalline multigelation for several times.Add 1 × PBS damping fluid (pH7.4) of precooling according to the twice of thalline weight in wet base volume, and add Triton-100 to final concentration be volume ratio 1%, with resuspended thalline.This bacteria suspension is put ultrasonic degradation cell in ice bath, power 400W, and ultrasonic 1s, leaves standstill 3s, and co-processing 10min is thickness extremely no longer.Through 4 ℃, the centrifugal 15min of 12000rpm, get respectively supernatant liquor and precipitation and carry out SDS-PAGE detection again.Collect supernatant liquor ,-20 ℃ save backup.
Preparing 1mL HisTrap HP column (is Ni
2+-NTA affinity column), with 10 times of column volume distilled water flushing pillars, by binding buffer liquid (40mmol/L imidazoles, 20mmol/L sodium radio-phosphate,P-32 solution, 500mmol/L NaCl, the pH7.4) balance of 10 times of column volumes.The above-mentioned supernatant samples through ultrasonic degradation is after 0.22 μ m filtering membrane is processed, with the flow velocity upper prop of about 1.0mL/min, wash post with the binding buffer liquid of 25 times of column volumes again, finally use elutriant (the 150mmol/L imidazoles of 10 times of column volumes, 20mmol/L sodium radio-phosphate,P-32 solution, 500mmol/L NaCl, pH7.4), wash-out object recombinant protein SjLys-N, the fusion rotein component of collecting sea cucumber antalzyme polypeptide.Albumen is concentrated into 8～10mg/mL by the Millipore ultra-filtration membrane that is 5kDa with molecular weight cut-off again.Concentrating sample, through the desalination of Sephadex G-25 gel chromatography, is collected to fusion rotein component, and lyophilize, obtains respectively the purified product (swimming lane 5 in Fig. 3) of recombinant sea cucumber antalzyme N end polypeptide.
The mensuration of embodiment 4 recombinant sea cucumber antalzyme N end polypeptide bacteriostatic activities
Adopt lysoplate assay to measure the inhibition zone size of recombinant sea cucumber antalzyme N end polypeptide to part gram-positive microorganism and Gram-negative bacteria, represent with its radius (mm).Some grams of lyophilized powders that first take SjLys-N, are dissolved in 1 × PBS (pH7.4), utilize content that Bradford method determines its albumen at 100 μ g/mL.The negative control of this experiment be empty carrier pET-32a (+) in E.coli Rosetta (DE3) pLysS, induce fermentation and purifying protein, be prepared into after lyophilized powder, more quantitatively to protein content be 100 μ g/mL.
Get gram-positive microorganism, streptococcus aureus (Staphylococcus aureus) and micrococcus lysodeikticus (Micrococcus lysodeikticus), and Gram-negative bacteria, intestinal bacteria K88 (Escherichia coli), Vibrio parahaemolyticus (Vibrio parahaemolyticus), Vibrio anguillarum (Vibrio anguillarium), Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Aeromonas hydrophila (Aeromonas hydrophila) are as indicator.By these bacteriums 37 ℃, 200rpm incubated overnight in LB liquid nutrient medium, be diluted to OD
600be 0.001 left and right, bacteria concentration is about 3.0 × 10
9cFU/ml.Get the above-mentioned selected bacterium liquid of 100 μ L and 20mL, the LB solid medium of 50 ℃ mixes rapidly, and it is cooling to pave plate, then tests as follows:
Test one: on the flat board that contains above-mentioned a certain specific indicator, places 2 aseptic Oxford cups, add respectively 100 μ L restructuring SjLys-N stostes and through 100 ℃, the restructuring SjLys-N solution of heating 40min processing.
Test two: on the flat board that contains above-mentioned a certain specific indicator, place 2 aseptic Oxford cups, add respectively 100 μ L restructuring SjLys-N stoste and negative control sample solutions.
The flat board of all tests is placed in to 30 ℃ of incubated overnight, measures inhibition zone.Test in triplicate results averaged.Table 1 is depicted as the inhibition zone size of this product after recombinant sea cucumber antalzyme N end polypeptide and heat treated, found that this recombinant sea cucumber antalzyme N end polypeptide all has restraining effect to Gram-negative bacteria and positive bacteria, especially gram negative pathogenic bacteria vibrios common in sea-food is had to obvious restraining effect, and SjLys-N after heated and inactivated remains unchanged substantially to the bacteriostasis of Gram-negative bacteria and positive bacteria, illustrate that this product has good thermostability, this is highly profitable as the application of fodder additives to sea cucumber antalzyme polypeptide.
The table 1 SjLys-N polypeptide Antibacterial Activity of recombinating
* use the purified extraction albumen of fermented liquid of negative control pET-32a (+)/E.coli Rosetta (DE3) pLysS, measure it to above-mentioned germy bacteriostatic activity, result does not all detect inhibition zone.
Claims (7)
1. the primer sequence of recombinant sea cucumber antalzyme N end polypeptide, is characterized in that: primer sequence is respectively forward primer: SEQ ID NO:1; Reverse primer: SEQ ID NO:2.
2. the plasmid of a recombinant sea cucumber antalzyme N end polypeptide, it is characterized in that, preparation method comprises: take N,O-Diacetylmuramidase SjLys gene as template, the gene fragment that primer sequence claimed in claim 1 is increased is connected in coli expression carrier pET32a (+), builds recombinant expression plasmid.
3. a genetic engineering bacterium of expressing recombinant sea cucumber antalzyme N end polypeptide, is characterized in that, preparation method comprises: plasmid claimed in claim 2 is transformed to Rosetta (DE3) pLysS host cell and obtain recombination engineering bacteria.
4. a preparation method for recombinant sea cucumber antalzyme N end polypeptide, is characterized in that, preparation method comprises:
1. abduction delivering: cultivate recombination engineering bacteria claimed in claim 3, and use IPTG abduction delivering;
2. separate and purifying: collect 1. products obtained therefrom of step, through nickel ion affinity chromatograph column purification, and concentrated with ultra-filtration membrane; To after concentrating sample desalination, make finished product again.
5. method according to claim 4, is characterized in that, the condition of abduction delivering and separation and purification comprises:
1. abduction delivering
By recombination engineering bacteria claimed in claim 3, in the liquid nutrient medium of optimizing, 37 ℃, 160rpm shaking culture are to OD
600reach 0.6-0.8, then be 0.5mmol/L to adding IPTG in culture system to final concentration, 28 ℃, 150rpm inducing culture 8h, collect fermented liquid;
Wherein, the liquid nutrient medium of optimization refers to: in LB substratum, add two kinds of microbiotic, i.e. and 100mg/L penbritin and 34mg/L paraxin, and add 5g/L glucose, 0.3g/L MgSO
47H
2o and 1g/L K
2hPO
43H
2o;
2. separate and purifying
The fermented liquid that 1. step is collected is centrifugal, collect and wash thalline, multigelation, with the resuspended thalline of phosphate buffered saline buffer, and after adding Triton X-100 to be 1% to final concentration, through the broken bacterium of ice-bath ultrasonic, centrifugal collection supernatant liquor, through nickel ion affinity chromatograph post His Trap HP column purifying, collect the fusion rotein component of recombinant sea cucumber antalzyme polypeptide, albumen is concentrated into 8～10mg/mL by the Millipore ultra-filtration membrane that is 5kDa with molecular weight cut-off; Again by concentrating sample through the desalination of Sephadex G-25 gel chromatography, collect fusion rotein component, lyophilize, obtains the product of recombinant sea cucumber antalzyme N end polypeptide.
6. the recombinant sea cucumber antalzyme N end polypeptide obtaining according to the method described in claim 4 or 5.
7. recombinant sea cucumber antalzyme N end polypeptide as claimed in claim 6 is in the application of preparing in fodder additives, food preservatives, antibiotic alternative medicine, sea farming production field.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210055509.7A CN102559672B (en) | 2012-03-05 | 2012-03-05 | Recombinant sea cucumber lysozyme N-terminal peptide, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210055509.7A CN102559672B (en) | 2012-03-05 | 2012-03-05 | Recombinant sea cucumber lysozyme N-terminal peptide, preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102559672A CN102559672A (en) | 2012-07-11 |
| CN102559672B true CN102559672B (en) | 2014-05-21 |
Family
ID=46406278
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210055509.7A Expired - Fee Related CN102559672B (en) | 2012-03-05 | 2012-03-05 | Recombinant sea cucumber lysozyme N-terminal peptide, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102559672B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586327B (en) * | 2014-10-21 | 2019-08-09 | 复旦大学 | A kind of source of people antalzyme protein purification process |
| CN110699269A (en) * | 2019-10-16 | 2020-01-17 | 东南大学 | Method for maintaining and culturing pichia pastoris transformant by using double antibiotics |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101423840A (en) * | 2008-12-15 | 2009-05-06 | 大连工业大学 | Method for producing recombinant sea cucumber antalzyme and recombinant sea cucumber antalzyme produced thereby |
-
2012
- 2012-03-05 CN CN201210055509.7A patent/CN102559672B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101423840A (en) * | 2008-12-15 | 2009-05-06 | 大连工业大学 | Method for producing recombinant sea cucumber antalzyme and recombinant sea cucumber antalzyme produced thereby |
Non-Patent Citations (3)
| Title |
|---|
| 周翔等.重组大鼠溶菌酶N端多肽的原核表达及其对晚期糖基化终产物的结合作用.《暨南大学学报（医学版）》.2006,第27卷(第6期),784-790. * |
| 海刺参i 型溶菌酶基因的重组表达及抑菌谱分析;王秀霞等;《生物工程学报》;20090225;第25卷(第2期);189-194 * |
| 王秀霞等.海刺参i 型溶菌酶基因的重组表达及抑菌谱分析.《生物工程学报》.2009,第25卷(第2期),189-194. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102559672A (en) | 2012-07-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102757947A (en) | Xylanase xyn-CDBFV-m with modified thermal stability, gene thereof, and application thereof | |
| CN101705212A (en) | N-acylhomoserine lactonas, production method thereof and special recombinant bacterium | |
| CN103275191B (en) | Method for largely and quick extracting tachyplesin peptide | |
| CN101104637B (en) | Cecropin and its generation bacterial strain and use | |
| CN101906165B (en) | Expression product in series of two fish antibacterial peptide genes and expression method thereof | |
| CN103382462B (en) | A kind of Bacillus licheniformis N,O-Diacetylmuramidase and production method thereof and application | |
| CN103789317A (en) | Scylla paramamosain antibacterial peptide hyastatin gene clone, encoding protein recombination and application | |
| CN104962572A (en) | Preparation method for aeromonas hydrophila outer membrane protein gene prokaryotic expression protein | |
| CN102731641A (en) | Modification of echinococcus granulosus EG95 protein, and expression thereof in yeast | |
| CN101423840B (en) | Method for producing recombinant sea cucumber antalzyme and recombinant sea cucumber antalzyme produced thereby | |
| CN102559672B (en) | Recombinant sea cucumber lysozyme N-terminal peptide, preparation method and application thereof | |
| CN104311645B (en) | Spirulina polypeptide P1 and its application with bacteriostatic activity | |
| CN103421707B (en) | Tibetan pig source bacillus and application thereof | |
| CN104263709A (en) | Egg-white lysozyme and preparation method thereof | |
| CN102242092A (en) | Isolation and identification for bombyx mori intestinal tract probiotics, and cloning and expression of enzyme gene produced by bombyx mori probiotics | |
| CN104961811A (en) | Aeromonas hydrophila outer membrane protein gene prokaryotic expression protein and application thereof | |
| CN109997970B (en) | Acidic xylanase mutant with improved enzyme activity and heat resistance, and coding gene and application thereof | |
| CN102586262B (en) | Defensin gene of antimicrobial peptide of bemisia tabaci (Gennadius), antimicrobial peptide encoded by defensin gene and preparation method for defensin gene | |
| CN102559671B (en) | Recombinant stichopus japonicus lysozyme C-terminal polypeptide and preparation method and application thereof | |
| CN103848912A (en) | Recombinant protein of bay scallop peptidoglycan recognition protein as well as preparation and application thereof | |
| CN102827854A (en) | Encoding gene of DON degrading enzyme and application thereof | |
| CN102978187A (en) | High-activity beta-mannanase MAN5A with pH value within range of 2.5-6.5, and gene and application thereof | |
| CN105567606A (en) | Arthrobacter globiformis and hyaluronidase generated by arthrobacter globiformis | |
| CN101565462B (en) | Large yellow croaker BPI-BN protein, primer and applications thereof in preparing antibiotic drugs | |
| CN106148381B (en) | A method of utilizing bacillus subtilis high efficient expression porcine defensin |
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: 20140521 Termination date: 20170305 |