CN112625986B - 一种高产表面活性素的基因工程菌及其构建方法和应用 - Google Patents
一种高产表面活性素的基因工程菌及其构建方法和应用 Download PDFInfo
- Publication number
- CN112625986B CN112625986B CN202011472978.XA CN202011472978A CN112625986B CN 112625986 B CN112625986 B CN 112625986B CN 202011472978 A CN202011472978 A CN 202011472978A CN 112625986 B CN112625986 B CN 112625986B
- Authority
- CN
- China
- Prior art keywords
- comq
- surfactant
- fermentation
- gene
- bacillus amyloliquefaciens
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000004094 surface-active agent Substances 0.000 title claims abstract description 52
- 241000894006 Bacteria Species 0.000 title claims abstract description 31
- 238000010276 construction Methods 0.000 title claims abstract description 19
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims abstract description 58
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 54
- 238000000855 fermentation Methods 0.000 claims abstract description 49
- 230000004151 fermentation Effects 0.000 claims abstract description 49
- 239000001963 growth medium Substances 0.000 claims abstract description 16
- 230000000415 inactivating effect Effects 0.000 claims abstract description 7
- NJGWOFRZMQRKHT-WGVNQGGSSA-N surfactin C Chemical compound CC(C)CCCCCCCCC[C@@H]1CC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)O1 NJGWOFRZMQRKHT-WGVNQGGSSA-N 0.000 claims description 23
- AFWTZXXDGQBIKW-UHFFFAOYSA-N C14 surfactin Natural products CCCCCCCCCCCC1CC(=O)NC(CCC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)O1 AFWTZXXDGQBIKW-UHFFFAOYSA-N 0.000 claims description 20
- NJGWOFRZMQRKHT-UHFFFAOYSA-N surfactin Natural products CC(C)CCCCCCCCCC1CC(=O)NC(CCC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)O1 NJGWOFRZMQRKHT-UHFFFAOYSA-N 0.000 claims description 20
- 239000013612 plasmid Substances 0.000 claims description 17
- 239000013600 plasmid vector Substances 0.000 claims description 17
- 239000012634 fragment Substances 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 108020004414 DNA Proteins 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 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
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 230000003213 activating effect Effects 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 230000002779 inactivation Effects 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 5
- 238000001976 enzyme digestion Methods 0.000 claims description 5
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 5
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 239000001301 oxygen Substances 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- 229940073490 sodium glutamate Drugs 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- 102000012410 DNA Ligases Human genes 0.000 claims description 4
- 108010061982 DNA Ligases Proteins 0.000 claims description 4
- 230000029087 digestion Effects 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 3
- 230000001131 transforming effect Effects 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 1
- 150000007523 nucleic acids Chemical group 0.000 claims 1
- 241000700605 Viruses Species 0.000 abstract description 14
- 238000010353 genetic engineering Methods 0.000 abstract description 14
- 206010012735 Diarrhoea Diseases 0.000 abstract description 9
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 2
- 229930027917 kanamycin Natural products 0.000 description 12
- 229960000318 kanamycin Drugs 0.000 description 12
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 12
- 229930182823 kanamycin A Natural products 0.000 description 12
- 241000193830 Bacillus <bacterium> Species 0.000 description 11
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 241000191967 Staphylococcus aureus Species 0.000 description 8
- 230000003385 bacteriostatic effect Effects 0.000 description 8
- 230000001276 controlling effect Effects 0.000 description 8
- 230000002949 hemolytic effect Effects 0.000 description 8
- 230000002401 inhibitory effect Effects 0.000 description 8
- 238000012258 culturing Methods 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
- 238000003780 insertion Methods 0.000 description 6
- 230000037431 insertion Effects 0.000 description 6
- 239000012880 LB liquid culture medium Substances 0.000 description 5
- 241001135549 Porcine epidemic diarrhea virus Species 0.000 description 5
- 108020005091 Replication Origin Proteins 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 229960003276 erythromycin Drugs 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 229960005091 chloramphenicol Drugs 0.000 description 4
- 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 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- 108010028921 Lipopeptides Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000002744 homologous recombination Methods 0.000 description 3
- 230000006801 homologous recombination Effects 0.000 description 3
- 238000007852 inverse PCR Methods 0.000 description 3
- 231100000219 mutagenic Toxicity 0.000 description 3
- 230000003505 mutagenic effect Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- 241000194108 Bacillus licheniformis Species 0.000 description 2
- 241001249117 Bacillus mojavensis Species 0.000 description 2
- 241000194103 Bacillus pumilus Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 230000032770 biofilm formation Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- PBEDWPIWZYHNPL-RHKAOURMSA-N surfactin A Chemical compound CC(C)CCCCCCC[C@@H]1CC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)O1 PBEDWPIWZYHNPL-RHKAOURMSA-N 0.000 description 2
- 108700038523 surfactin A Proteins 0.000 description 2
- DDULCZLFAKVKJB-JIXKVBNCSA-N surfactin B Chemical compound CC(C)CCCCCCCC[C@@H]1CC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)O1 DDULCZLFAKVKJB-JIXKVBNCSA-N 0.000 description 2
- 230000002537 thrombolytic effect Effects 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000013566 Plasminogen Human genes 0.000 description 1
- 108010051456 Plasminogen Proteins 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000002832 anti-viral assay Methods 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000003876 biosurfactant Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- -1 carbon atom fatty acid Chemical class 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000010227 cup method (microbiological evaluation) Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- XQGPKZUNMMFTAL-UHFFFAOYSA-L dipotassium;hydrogen phosphate;trihydrate Chemical compound O.O.O.[K+].[K+].OP([O-])([O-])=O XQGPKZUNMMFTAL-UHFFFAOYSA-L 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 231100000405 induce cancer Toxicity 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000004779 membrane envelope Anatomy 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 108010073863 saruplase Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000003501 vero cell Anatomy 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- AFVLVVWMAFSXCK-UHFFFAOYSA-N α-cyano-4-hydroxycinnamic acid Chemical class OC(=O)C(C#N)=CC1=CC=C(O)C=C1 AFVLVVWMAFSXCK-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K11/00—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K11/02—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof cyclic, e.g. valinomycins ; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- General Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种高产表面活性素的基因工程菌及其构建方法和应用,所述基因工程菌以解淀粉芽孢杆菌CPLK1314为出发菌株,敲除或者失活菌株基因组中负调控ComQ基因所得。本发明失活ComQ的基因工程菌在优化的培养基和发酵条件下用于生产表面活性素产量是野生型菌株的5倍,在5L发酵罐40‑50h可稳定生产表面活性素5‑6g/L。该表面活性素高产基因工程菌及其构建方法为加速表面活性素的产业化提供原材料,同时高产表面活性素的基因工程菌可用于制备猪腹泻病毒的防治生物试剂。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种高产表面活性素的基因工程菌及其构建方法与应用。
背景技术
表面活性素(surfactins)是由7个氨基酸亲水性短肽头部和13~15碳原子的脂肪酸的疏水性尾部组成的一类双亲性环脂肽化合物,是目前已知最强的生物表面活性剂,具有抗细菌抗病毒,抗肿瘤,抑制生物膜形成和促进血栓溶解等生物活性。相对于传统的抗生素,表面活性素对多种耐药性细菌比如金黄色葡萄球菌具有更高的抑制活性和更低的细胞毒性。表面活性素通过渗透病毒包膜形成穿孔,对多种包膜病毒比如猪腹泻病毒具有很强的抗病毒活性。表面活性素能作为信号分子诱导癌细胞凋亡,从而表现出抗肿瘤的活性。生物膜是许多病原微生物产生耐药性和污染医疗器官的主要原因,表面活性素也展示出很强的抑制病原微生物比如黏附能力和生物膜形成的活性。表面活性素还能够激活尿激酶原导致血纤维蛋白溶酶原的构象改变,从而促进血栓的溶解。总之,表面活性素由于具有上述的生物学活性,在植物保护,食品安全和现代医疗中都具有广泛的用途。由于表面活性素特殊的大环内酯结构及含有多个手性基团,采用化学合成的方法存在较大的难度,因此目前生产表面活性素的生产方法主要是通过芽孢杆菌发酵的方法制备。
合成分泌表面活性素的菌株主要有枯草芽孢杆菌(B.subtilis),解淀粉芽孢杆菌(B.amyloliquefaciens),地衣芽孢杆菌(B.licheniformis),莫加芽孢杆菌(B.mojavensis)和短小芽孢杆菌(B.pumilus)等。不同的芽孢杆菌合成分泌不同的表面活性素异构体,绝大多数芽孢杆菌菌株合成分泌量低于1.0g/l,严重限制了表面活性素进入生产实践应用,通过基因工程的方法提高野生型芽孢杆菌表面活性素的产量是目前研究的一个热点。
发明内容
发明目的:针对现有技术存在的问题,本发明提供一种高产表面活性素的解淀粉芽孢杆菌基因工程菌,本发明通过基因工程手段构建出高产表面活性素的基因工程菌株,并制定了相应的发酵过程控制方案,该方法可应用于表面活性素的工业化生产。
本发明还提供高产表面活性素的基因工程菌及其构建方法与应用。
技术方案:为了实现上述目的,本发明所述一种高产表面活性素的基因工程菌,所述基因工程菌以解淀粉芽孢杆菌为出发菌株,敲除或者失活菌株基因组中负调控ComQ基因所得。
其中,所述负调控ComQ基因的核苷酸序列如SEQ ID NO:1所示。
作为优选,所述解淀粉芽孢杆菌为解淀粉芽孢杆菌CPLK1314,保藏号CCTCC NO:M2017658。
本发明所述的负调控核苷酸序列ComQ基因,是经过转座子pMarA插入,突变体库筛选和插入位点分析获得。
本发明所述高产表面活性素的基因工程菌的构建方法,包括如下步骤:
(1)以ComQ基因序列设计引物,以解淀粉芽孢杆菌的基因组DNA作为模板,扩增到部分ComQ基因片段;
(2)同源重组质粒载体pTCCOMQ的构建:扩增的ComQ基因片段经过XhoⅠ和EcoRⅠ双酶切,然后经过T4DNA连接酶连接到经过同样双酶切的质粒载体pTC中,构建同源重组整合质粒载体pTCCOMQ;
(3)ComQ基因失活突变菌株ΔComQ的构建:将构建好的重组质粒pTCCOMQ转化至解淀粉芽孢杆菌得到高产表面活性素的基因工程菌ComQ基因突变株ΔComQ。
其中,步骤(1)所述的引物为:ComQ-F COMQF(5′-TTTGAATTCGGGATTCATATAGCTAAGAA-3′)和COMQR(5′-TTTCTCGAGAAATAAATCATTTTTTAATTG-3′)。
其中,所述重组质粒载体pCOMQ,该重组质粒载体含有ComQ基因同源双交换臂,卡那霉素抗性基因,可定向对解淀粉芽孢杆菌(B.amyloliquefaciens)CPLK1314中得ComQ基因进行失活。
本发明所述高产表面活性素的基因工程菌在发酵生产表面活性素中的应用。
其中,所述发酵为通过基因工程菌进行发酵罐发酵:将菌种活化后制备种子液,按照体积比10-20%接种量接入新鲜的发酵培养基,开始发酵,发酵过程中控制pH稳定在6.5左右,温度维持在30℃,溶氧在25-35%之间;发酵周期48-50h。
其中,所述发酵培养基组成为:葡萄糖10-15g/L,蔗糖10-15g/L,酵母粉5-8g/L,谷氨酸钠5-6g/L,蛋白胨1-5g/L,K2HPO4 1g/L,KH2PO4 1g/L,MgSO4·7H2O 500mg/L,FeSO4·7H2O 10mg/L,MnSO4·H2O 10mg/L,VB1、VB3、VB5、VB12、VH各1-3mg/L,其余为水,pH 7.0。
该发酵培养基和发酵条件可以提升所述高产工程菌中表面活性素的产量达到5-6g/L。
本发明所述高产表面活性素的基因工程菌在制备用于猪腹泻病毒的防治生物试剂中的应用。
本发明所述ComQ基因失活的解淀粉芽孢杆菌(B.amyloliquefaciens)CPLK1314生产的表面活性素对猪腹泻病毒具有很强的钝化和抑制病毒繁殖的作用,可应用猪腹泻病毒的防治。
本发明所述ComQ编码基因在调控解淀粉芽孢杆菌表面活性素产量中的应用。
本发明提供了一种负调控表面活性素合成的基因ComQ,ComQ是通过构建解淀粉芽孢杆菌(B.amyloliquefaciens)CPLK1314转座子(pMarA)突变体库,以金黄色葡萄球菌作为指示菌,根据抑菌活性变化筛选突变株;然后采用反向PCR克隆侧翼序列鉴定获得。
本发明所述解淀粉芽孢杆菌CPLK1314突变体库的构建,包括如下步骤:
(1)将转座子pMarA质粒转化到解淀粉芽孢杆菌中,并利用卡那霉素和红霉素双抗LB平板培养;
(2)将步骤(1)含有pMarA质粒的转化子接种至含有卡那霉素和红霉素的双抗LB液体培养基中,重复转接活化后,转接入只含卡那霉素的LB液体中,培养;离心收集的细胞为构建的突变体库;
(3)将突变体均匀涂布在含有卡那霉素抗性平板上,从抗性平板上挑取单菌落点在含有金黄色葡萄球菌的指示平板上,培养后,测量抑菌圈的大小,都得具有抑菌效果较好的突变菌株。
本发明所述高产基因工程菌合成表面活性素过程中主要调控基因的鉴定过程。
以获得解淀粉芽孢杆菌CPLK1314突变株插入位点侧翼序列克隆及分析。将突变株接种于卡那霉素的LB液体培养基中,培养,采用全式金生物公司的基因组DNA提取试剂盒提取突变株H5基因组DNA。根据转座子pMarA序列设计IPCR1(5'-GCTTGTAAATTCTATCATAATTG-3')和IPCR2(5'-AGGGAATCATTTGAAGGTTGG-3')引物对,以突变株基因组DNA作为模板PCR扩增,经测序和在NCBI进行同源分析发现突变株转座子插入位点为ComQ基因,核苷酸序列见SEQ ID NO.1。
相对于传统的物理化学诱变,诱变菌株不稳定,诱变位点不容易分析的缺点,本发明采用转座子诱变芽孢杆菌结合抑菌圈平板指示的方法,筛选到高产表面活性素的突变株,并通过反向PCR克隆负调控表面活性素合成的基因ComQ和通过同源重组的方法获得负调控基因破坏的高产表面活性素的突变株ΔComQ。该突变株从抑菌活性,溶血活性都显著由于出发菌株;通过该菌株制备的表面活性素对猪腹泻病毒也有显著抑制活性。
本发明通过转座子pMarA插入解淀粉芽孢杆菌CPLK1314鉴定到一个负调控表面活性素合成的因子ComQ,ComQ基因的失活能显著提高解淀粉芽孢杆CPLK1314合成表面活性素的能力;并提供了一种高产表面活性素的发酵培养和发酵条件,表面活性素合成的产量达到5-6g/L。
有益效果:与现有技术相比,本发明具有如下优点:
本发明通过对淀粉芽孢杆菌CPLK1314基因组中负调控ComQ基因敲除或者失活构建出高产表面活性素的基因工程菌,该基因工程菌株与野生型解淀粉芽孢杆菌CPLK1314相比表面活性素的产量提高了5倍,在优化后的培养基中每升发酵液可制备获得5-6g/L表面活性素,该基因工程菌生产的表面活性素可应用于用于猪腹泻病毒的防治,本发明的基因工程菌及其构建方法为加速表面活性素的产业化提供原材料,同时高产表面活性素的基因工程菌可用于制备猪腹泻病毒的防治生物试剂。
附图说明
图1为反向PCR克隆转座子pMarA插入位点ComQ示意图;
图2为用于失活ComQ基因的同源重组质粒载体pTCCOMQ示意图;
图3为解淀粉芽孢杆菌CPLK1314和ComQ基因失活突变株ΔComQ产表面活性素高效液相色谱分析示意图;
图4为表面活性素的质谱检测示意图;
图5为解淀粉芽孢杆菌CPLK1314和ComQ基因失活突变株ΔComQ抑制金黄色葡萄球菌活性示意图;
图6为解淀粉芽孢杆菌CPLK1314和ComQ基因失活突变株ΔComQ溶血活性示意图。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
本发明实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂家建议的条件。其中淀粉芽孢杆菌CPLK1314,保藏号CCTCC NO:M2017658,由淮阴工学院提供。
转座子pMarA质粒和pSG1164质粒来源于美国芽孢杆菌菌株保藏中心(BacillusGenetic Stock Center),货号分别为ECE191(参考文献:Le breton Y,et al.(2006)ApplEnviron Microbiol 72:327)和ECE155(参考文献:Lewis PJ and Marston AL.(1999)Gene227:101)。
pUC19质粒载体采用常规市售均可。
质粒载体pTC由淮阴工学院构建保藏,含有来源pUC19(TranGene)的大肠杆菌复制起点和氨苄青霉素抗性基因,来源于pMarA的芽孢杆菌热敏复制起点,来源于pSG1164氯霉素抗性基因和含有XhoⅠ和EcoRⅠ酶切位点的多克隆位点,具体构建过程如实施例3。
解淀粉芽孢杆菌CPLK1314感受态细胞制备相关试剂配制:Sp-A(0.4g硫酸铵,0.2g磷酸二氢钾,3.66g三水磷酸氢二钾,加水至100ml);Sp-B(0.04g硫酸镁,加水至100mL),CAYE(0.1g Casamino acid,0.5g酵母提取物,加水至5ml);SpI(Sp-A和Sp-B各98ml,2ml50%葡萄糖,2ml CAYE);SpII(588ml SpI,0.6ml 50mmol/L CaCl2,0.6ml 250mmol/LMgCl2)。
实施例1
解淀粉芽孢杆菌(B.amyloliquefaciens)CPLK1314突变体库的构建及筛选
转座子pMarA质粒转化至解淀粉芽孢杆菌CPLK1314感受态细胞中:挑单菌落接种至25ml SpI培养基,30℃、180r/min振荡培养过夜。以1:25体积接种到新鲜的SpI培养基,30℃、250r/min振荡培养OD600至2.0,约3.5h;以1:10体积接种到SpII培养基,37℃、150r/min振荡培养1.5h后,以5000r/min离心收集。菌体以上清液1/10体积进行悬浮即用作感受态细胞。在250μL感受态细胞中加入终浓度为1mmol/L EGTA,37℃,150r/min振荡培养,10min后加入1μg pMarA质粒,37℃,150r/min振荡培养1h,再以37℃,250r/min继续振荡培养30min。最后涂布在含有卡那霉素(50μg/mL)和红霉素(1μg/mL)的LB双抗性平板上。
转座子突变体库构建及突变体库筛选:将含有pMarA质粒的转化子(挑取上述双抗LB平板上单菌落)接种至含有卡那霉素(50μg/mL)和红霉素(1μg/mL)的LB液体培养基30℃,150r/min培养过夜,取1mL液体加入到25mL含有5μg/mL卡那霉素、1μg/mL红霉素双抗LB液体三角瓶里面,30℃,150r/min培养到待其OD值达到0.5-0.6之后再次从中取出1mL液体加入到100mL只含5μg/mL卡那霉素的LB液体三角瓶,50℃200r/min继续振荡培养12h;离心收集的细胞为构建的突变体库。将突变体均匀涂布在含有50μg/mL卡那霉素抗性平板上,从抗性平板上挑取单菌落点在含有金黄色葡萄球菌的指示平板上,放置于28℃下培养,得到与解淀粉芽孢杆菌CPLK1314抑菌圈对比抑菌效果显著提高的突变体H5。
实施例2
解淀粉芽孢杆菌CPLK1314突变株H5插入位点侧翼序列克隆及分析。
将突变株H5接种于含5μg/mL卡那霉素的LB液体培养基中,30℃,180r/min培养24h,采用全式金生物公司的基因组DNA提取试剂盒提取突变株H5基因组DNA。根据转座子pMarA序列设计引物,以IPCR1(5'-GCTTGTAAATTCTATCATAATTG-3')和IPCR2(5'-AGGGAATCATTTGAAGGTTGG-3')作为引物对,以经过Sau3A不完全酶切和T4 DNA连接酶环化的H5突变株基因组DNA作为模板;扩增体系如表1。PCR扩增程序:95℃3min;95℃30s;55℃45s;72℃40s,30个循环;72℃10min;4℃保存;扩增到一条约500bp条带,核酸凝胶电泳结果如图1,经测序和在NCBI进行同源分析发现突变株H5转座子插入位点为ComQ基因,核苷酸序列见SEQ ID NO.1。
表1反向PCR反应体系
组成 | 体积 |
10XPCR缓冲液 | 5μl |
100μg/ml环化突变株模板基因组DNA | 1μl |
20mM IPCR1引物 | 1μl |
20mM IPCR2引物 | 1μl |
2.5mM dNTPs | 4μl |
<sub>EX</sub> Taq DNA polymerase | 1μl |
dd H<sub>2</sub>O | 37μl |
实施例3
用于失活解淀粉芽孢杆菌CPLK1314中ComQ基因同源重组质粒载体pTCCOMQ的构建及ComQ突变株ΔComQ的构建
首先构建大肠杆菌和芽孢杆菌穿梭质粒pTC,构建的引物如表2所示。
表2用于构建的pTC质粒的引物
采用PCR反应体系和反应条件如实施例2。具体过程如下,采用pTCCF和pTCCR引物对,以pSG1164为模板扩增到氯霉素抗性基因,扩增基因片段经过EcoRI和SmaI双酶切后插入到经过同样双酶切的pUC19质粒载体构建为pUC19-Cl;以pTCMF和pTCMR作为引物对,以pMarA质粒作为模板,扩增到芽孢杆菌热敏复制起点,扩增基因片段经过SmaI和PstI双酶切后插入pUC19-Cl构建为pUC19-Cl-OR;采用pTCCF和pTCMR作为引物对,以pUC19-Cl-OR作为模板扩增融合氯霉素抗性基因和芽孢杆菌热敏复制起点的融合基因片段;采用pTC19F和pTC19R扩增带有氨苄青霉素和大肠杆菌复制起点的基因片段;将扩增的两个基因片段经过EcoRI和KpnI双酶切后,采用T4DNA连接酶连接,构建穿梭质粒载体pTC,核苷酸序列如SEQID NO.2所示。
带有ComQ同源交换片段和氯霉素抗性基因的同源重组质粒载体pTCCOMQ构建过程如下:根据ComQ部分基因序列设计COMQ-F(5′-TTTGAATTCGGGATTCATATAGCTAAGAA-3′)和COMQ-R(5′-TTTCTCGAGAAATAAATCATTTTTTAATTG-3′)引物对分别如序列SEQ ID NO.3-4所示,以解淀粉芽孢杆菌CPLK1314的基因组DNA作为模板,PCR扩增到一条长约600bp的部分ComQ基因片段;扩增的基因片段经过XhoⅠ和EcoRⅠ双酶切(表2),然后经过割胶纯化和T4DNA连接酶链接(表3)到经过同样双酶切的质粒载体pTC,构建同源重组整合质粒载体pTCCOMQ.质粒pTCCOMQ质粒图谱见图2和核苷酸序列见SEQ ID NO.5。然后经过实施例1的方法将质粒pTCCOMQ转化至解淀粉芽孢杆菌CPLK1314,得到ComQ基因突变株ΔComQ。
表3酶切体系
组成 | 体积 |
10X酶切缓冲液 | 5μl |
100μg/ml质粒或PCR片段 | 35μl |
XhoⅠ限制性内切酶 | 5μl |
EcoRⅠ限制性内切酶 | 5μl |
表4连体系
实施例4
解淀粉芽孢杆菌CPLK1314和突变株ΔComQ发酵产表面活性素的定性定量分析。
从平板挑取解淀粉芽孢杆菌CPLK1314和突变株ΔComQ分别接种在250mL LB液体培养基中30℃,180rpm活化24h,活化后的菌株按体积比10%接种量接入5L发酵罐,含有新鲜的LB发酵培养基(10g/L胰蛋白,5g/L酵母粉,5g/LNaCl)或优化的培养基(葡萄糖10g/L,蔗糖10g/L,酵母粉5g/L,5g/L谷氨酸钠,蛋白胨2.5g/L,K2HPO4 1g/L,KH2PO4 1g/L,MgSO4·7H2O 500mg/L,FeSO4·7H2O 10mg/L,MnSO4·H2O 10mg/L,VB1、VB3、VB5、VB12、VH各2mg/L,其余为水,pH 7.0)开始发酵,发酵过程中控制pH值在6.5,温度维持在30℃,溶氧在30%左右,发酵48小时;通过10000r/min速度离心10min获得除菌体之外的5L发酵液上清液,用浓盐酸调节pH值到2,静置过夜进行表面活性素的沉淀。通过12000r/min速度离心20min获得沉淀,用50mL纯甲醇溶液溶解沉淀,用直径是0.22μm的过滤器进行过滤除去大颗粒杂质制备初提的表面活性素。
表面活性素用于高效液相色谱HPLC的检测条件为:安捷伦1200系列高效液相色谱HPLC和C18柱(5μm,4mm×250mm;Merck,Frankfurt,Germany),流动相为(乙腈:水:三氟乙酸(20:80:0.5(V/V)),检测波长均为210nm,流速为0.8mL/min,柱温是30℃。解淀粉芽孢杆菌CPLK1314和突变株ΔComQ发酵产表面活性素的量见图3和表5。
表5解淀粉芽孢杆菌CPLK1314和突变株ΔComQ发酵产表面活性素
由图3和表5可以看出,本发明构建的突变株ΔComQ发酵后可产生大量的表面活性素,并且明显优于原始的解淀粉芽孢杆菌CPLK1314。此外,采用本发明优化后的培养基,其表面活性素产量可以进一步提高。
表面活性素的质谱检测条件:质谱分析的仪器为Agilent 6410三重串联四级杆质谱仪,购自美国安捷伦科技股份有限公司。按照粗制备脂肽类化合物,采用含有饱和α-氰基-4-羟基桂皮酸,0.1%三氟乙酸,乙腈和水(体积比3﹕1)溶液稀释1 000倍后,通过注射器直接进样。采用质谱法测定粗提脂肽类化合物的分子质量,电喷雾条件为:毛细管电压32V、喷雾电压20kV、毛细管温度320℃。检测方式:正离子。表面活性素的质谱检测结果见图4和表6。
表6表面活性素的质谱检测分析
由图4和表6可以看出从解淀粉芽孢杆菌CPLK1314和突变株ΔComQ成功制备到表面活性素A,B和C(surfactin A,B和C)三种相差一个亚甲基(-CH2)同系物,同系物的分子量分别为1007.6,1021.7和1035.7。
实施例5
实施例5与实施例4的发酵方法相同,不同之处在于:将菌种活化后制备种子液,按照体积比20%接种量接入新鲜的发酵培养基,开始发酵,发酵过程中控制pH稳定在6.5左右,温度维持在30℃,溶氧在25%之间;发酵周期40h。发酵培养基组成为:葡萄糖15g/L,蔗糖15g/L,酵母粉8g/L,谷氨酸钠6g/L,蛋白胨5g/L,K2HPO4 1g/L,KH2PO4 1g/L,MgSO4·7H2O500mg/L,FeSO4·7H2O 10mg/L,MnSO4·H2O 10mg/L,VB1、VB3、VB5、VB12、VH各3mg/L,其余为水,pH 7.0。
实施例6
实施例6与实施例4的发酵方法相同,不同之处在于:将菌种活化后制备种子液,按照体积比15%接种量接入新鲜的发酵培养基,开始发酵,发酵过程中控制pH稳定在6.5左右,温度维持在30℃,溶氧在35%之间;发酵周期40h。发酵培养基组成为:葡萄糖12g/L,蔗糖12g/L,酵母粉6g/L,谷氨酸钠5g/L,蛋白胨1g/L,K2HPO4 1g/L,KH2PO4 1g/L,MgSO4·7H2O500mg/L,FeSO4·7H2O10mg/L,MnSO4·H2O 10mg/L,VB1、VB3、VB5、VB12、VH各1mg/L,其余为水,pH 7.0。
实施例7
解淀粉芽孢杆菌CPLK1314和变株ΔComQ抑制金黄色葡萄球菌分析。
采用牛津杯法对解淀粉芽孢杆菌CPLK1314和变株ΔComQ抑制金黄色葡萄球菌的抑菌活性进行测定,具体步骤包括:挑取金黄色葡萄球菌单菌落接于5mL LB液体培养基中,30℃,180rpm/min条件下培养12h。按体积比2%的量将病原菌菌液接种至50mL冷却至40℃的LB固体培养基中,混匀后倒入无菌平板中,将平板三等分并做好标注,无菌操作下夹取3个牛津杯垂直放置在平板上,在LB培养基和优化培养基培养的解淀粉芽孢杆菌CPLK1314和变株ΔComQ发酵上清液(制备同实施例4)各取150uL打入到牛津杯中,进行实验对比,静置平放于30℃培养箱中培养24h,观察牛津杯周围抑菌圈,抑菌圈直径(mm)=测量总直径(mm)-牛津杯直径(mm)。突变株ΔComQ发酵上清液抑菌效果明显优于解淀粉芽孢杆菌CPLK1314,结果如图5。
实施例8
解淀粉芽孢杆菌CPLK1314和变株ΔComQ溶血活性。
将解淀粉芽孢杆菌CPLK1314和变株ΔComQ均匀划线在含有5%的新鲜的绵羊血红细胞的琼脂培养基上,30℃培养1~3d,观察划线菌落周围的溶血透明圈,比较解淀粉芽孢杆菌CPLK1314和突变株ΔComQ溶血活性。解淀粉芽孢杆菌CPLK1314和变株ΔComQ的溶血活性见图6,由图6可以看出变株ΔComQ相对于解淀粉芽孢杆菌CPLK1314更早出现溶血活性,在48小时后其溶血活性圈也更明亮,说明突变株ΔComQ相对于解淀粉芽孢杆菌CPLK1314合成表面活性素时间早,能力强。
实施例9
解淀粉芽孢杆菌生产表面活性素对猪腹泻病毒的抑制活性。
采用实施例4突变株ΔComQ制备的表面活性素进行对猪流行性腹泻病毒(PEDV)进行抗病毒检测:待24孔板中的Vero细胞长满单层后,接种0.01MOI的PEDV,病毒感作1小时后换维持液,其中实验组中加入100μg/ml,50μg/ml,10μg/ml和5μg/ml浓度的表面活性素,37℃下培养36小时,收取细胞样品,提取病毒RNA,荧光定量方法检测PEDV含量。结果表明表面活性素在10μg/ml以上时PEDV病毒含量下降200倍以上,显示出很强的抗PEDV活性。
序列表
<110> 淮阴工学院
江苏省农业科学院
<120> 一种高产表面活性素的基因工程菌及其构建方法和应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 942
<212> DNA
<213> ComQ基因(ComQ)
<400> 1
gggattcata tagctaagaa attaactcta gaagcagaag atgtaaagga cacaataaac 60
catattctca aaaaaaatga tgtgaataaa gaagtaagta aacatgttgt gaattttgct 120
gatacaaaaa acaactttcc atttgctgag ttagctttag atcattattt ggcttttgga 180
gggaatgatt ttgaagaaat actagattta ggaacaggga ttgaactgct tatcttagcg 240
gctgatattt ttgatgacat acaagataag gatcgtccaa atgcagaatg gatgaaaatg 300
gatcaggctg tatctttaaa tattgcaacc ttaatttata caatagctct ccaagttata 360
tcatctcgag atccggaagg attattactc aagacagctc taaaatatac agttcaagct 420
atggaagggc aacacattga tttggttgat aatactttaa ctgaaaatca atgcattgaa 480
atgattaaga aaaagtcagg ggctttgaca gctttagcaa gtatgctggg tgctgtactt 540
gcaactaaac gttatcaatc agatgtggag cagtatgctt tctatatagg ggttgcagct 600
caattaaaaa atgatttatt tgagataatg tatgtgaata atacggaaac ctttttcaaa 660
aagacaaatt tggcaattca gttcttagag aaaaaattca acacagtatc cacagaattg 720
ttaaacaaca actttcaaca atcagagtta aaacggcttc tcttagactc tggtgttatc 780
cattatatgt cagtgatgat taatgtatat aatttgaaag tgaaaaatgg actcgaaaat 840
ttaaatttga ctttagagaa caaaaactat ttaattaaaa aaatttgcga aaatgagaag 900
gtggataaaa atgcaggaaa tagtaaatta tttagttcgt aa 942
<210> 2
<211> 5633
<212> DNA
<213> 质粒载体pTC(pTC)
<400> 2
gaattcacta gtgggcccag atctctcgag ttaagccagc cccgacaccc gccaacaccc 60
gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc 120
gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga 180
aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag 240
acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa 300
atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataataa 360
aaaaggattg attctaatga agaaagcaga caagtaagcc tcctaaattc actttagata 420
aaaatttagg aggcatatca aatgaacttt aataaaattg atttagacaa ttggaagaga 480
aaagagatat ttaatcatta tttgaaccaa caaacgactt ttagtataac cacagaaatt 540
gatattagtg ttttataccg aaacataaaa caagaaggat ataaatttta ccctgcattt 600
attttcttag tgacaagggt gataaactca aatacagctt ttagaactgg ttacaatagc 660
gacggagagt taggttattg ggataagtta gagccacttt atacaatttt tgatggtgta 720
tctaaaacat tctctggtat ttggactcct gtaaagaatg acttcaaaga gttttatgat 780
ttataccttt ctgatgtaga gaaatataat ggttcgggga aattgtttcc caaaacacct 840
atacctgaaa atgctttttc tctttctatt attccatgga cttcatttac tgggtttaac 900
ttaaatatca ataataatag taattacctt ctacccatta ttacagcagg aaaattcatt 960
aataaaggta attcaatata tttaccgcta tctttacagg tacatcattc tgtttgtgat 1020
ggttatcatg caggattgtt tatgaactct attcaggaat tgtcagatag gcctaatgac 1080
tggcttttat aatatgagat aatgccgact gtacttttta cagtcggttt tctaatgtca 1140
ctaacctgcc ccgttagttg aagaaggttt ttatattaca gctccagatc catatccttc 1200
tttttctgaa ccgacttctc ctttttcgct tctttattcc aattgcttta ttgacgttga 1260
gcctcggaac ccttaacaat cccaaaactt gtcgaatggt cggcttaata gctcacgcta 1320
tgccgacatt cgtctgcaag tttagttaag ggttcttctc aacgcacaat aaattttctc 1380
ggcataaatg cgtggtctaa tttttatttt taataacctt gatagcaaaa aatgccattc 1440
caatacaaaa ccacatacct ataatcgata accacataac agtcataaaa ccactccttt 1500
ttaacaaact ttatcacaag aaatatttac ccggggtcca gaaggtcgat agaaagcgtg 1560
agaaacagcg tacagacgat ttagagatgt agaggtactt ttatgccgag aaaacttttt 1620
gcgtgtgaca gtccttaaaa tatacttaga gcgtaagcga aagtagtagc gacagctatt 1680
aactttcggt tgcaaagctc taggattttt aatggacgca gcgcatcaca cgcaaaaagg 1740
aaattggaat aaatgcgaaa tttgagatgt taattaaaga cctttttgag gtcttttttt 1800
cttagatttt tggggttatt taggggagaa aacatagggg ggtactacga cctcccccct 1860
aggtgtccat tgtccattgt ccaaacaaat aaataaatat tgggttttta atgttaaaag 1920
gttgtttttt atgttaaagt gaaaaaaaca gatgttggga ggtacagtga tggttgtaga 1980
tagaaaagaa gagaaaaaag ttgctgttac tttaagactt acaacagaag aaaatgagat 2040
attaaataga atcaaagaaa aatataatat tagcaaatca gatgcaaccg gtattctaat 2100
aaaaaaatat gcaaaggagg aatacggtgc attttaaaca aaaaaagata gacagcactg 2160
gcatgctgcc tatctatgac taaattttgt taagtgtatt agcaccgtta ttatatcatg 2220
agcgaaaatg taataaaaga aactgaaaac aagaaaaatt caagaggacg taattggaca 2280
tttgttttat atccagaatc agcaaaagcc gagtggttag agtatttaaa agagttacac 2340
attcaatttg tagtgtctcc attacatgat agggatactg atacagaagg taggatgaaa 2400
aaagagcatt atcatattct agtgatgtat gagggtaata aatcttatga acagataaaa 2460
ataattaaca gaagaattga atgcgactat tccgcagatt gcaggaagtg tgaaaggtct 2520
tgtgagatat atgcttcaca tggacgatcc taataaattt aaatatcaaa aagaagatat 2580
gatagtttat ggcggtgtag atgttgatga attattaaag aaaacaacaa cagatagata 2640
taaattaatt aaagaaatga ttgagtttat tgatgaacaa ggaatcgtag aatttaagag 2700
tttaatggat tatgcaatga agtttaaatt tgatgattgg ttcccgcttt tatgtgataa 2760
ctcggcgtat gttattcaag aatatataaa atcaaatcgg tataaatctg accgatagat 2820
tttgaattta ggtgtcacaa gacactcttt tttcgcacca gcgaaaactg gtttaagccg 2880
actgcgcaaa agacataatc gattcacaaa aaataggcac acgaaaaaca agttaaggga 2940
tgcagtttat gcatccctta acggtaccac tggccgtcgt tttacaacgt cgtgactggg 3000
aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc 3060
gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 3120
aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat 3180
ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc 3240
caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag 3300
ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg 3360
cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg 3420
tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 3480
ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 3540
aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct 3600
tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag 3660
atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta 3720
agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc 3780
tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca 3840
tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg 3900
atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg 3960
ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca 4020
tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa 4080
acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa 4140
ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata 4200
aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat 4260
ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc 4320
cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata 4380
gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt 4440
actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga 4500
agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 4560
cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 4620
tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 4680
agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 4740
ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 4800
acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 4860
ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 4920
gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 4980
gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 5040
gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 5100
tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 5160
caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 5220
tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 5280
gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 5340
agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt 5400
ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 5460
gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 5520
ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 5580
atgaccatga ttacgccaag cttgcatgcc cgcggggatc cgctagctct aga 5633
<210> 3
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tttgaattcg ggattcatat agctaagaa 29
<210> 4
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tttctcgaga aataaatcat tttttaattg 30
<210> 5
<211> 6236
<212> DNA
<213> 质粒pTCCOMQ(pTCCOMQ)
<400> 5
gaattcggga ttcatatagc taagaaatta actctagaag cagaagatgt aaaggacaca 60
ataaaccata ttctcaaaaa aaatgatgtg aataaagaag taagtaaaca tgttgtgaat 120
tttgctgata caaaaaacaa ctttccattt gctgagttag ctttagatca ttatttggct 180
tttggaggga atgattttga agaaatacta gatttaggaa cagggattga actgcttatc 240
ttagcggctg atatttttga tgacatacaa gataaggatc gtccaaatgc agaatggatg 300
aaaatggatc aggctgtatc tttaaatatt gcaaccttaa tttatacaat agctctccaa 360
gttatatcat ctcgagatcc ggaaggatta ttactcaaga cagctctaaa atatacagtt 420
caagctatgg aagggcaaca cattgatttg gttgataata ctttaactga aaatcaatgc 480
attgaaatga ttaagaaaaa gtcaggggct ttgacagctt tagcaagtat gctgggtgct 540
gtacttgcaa ctaaacgtta tcaatcagat gtggagcagt atgctttcta tataggggtt 600
gcagctcaat taaaaaatga tttatttctc gagttaagcc agccccgaca cccgccaaca 660
cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 720
accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga 780
cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 840
tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 900
taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 960
taaaaaagga ttgattctaa tgaagaaagc agacaagtaa gcctcctaaa ttcactttag 1020
ataaaaattt aggaggcata tcaaatgaac tttaataaaa ttgatttaga caattggaag 1080
agaaaagaga tatttaatca ttatttgaac caacaaacga cttttagtat aaccacagaa 1140
attgatatta gtgttttata ccgaaacata aaacaagaag gatataaatt ttaccctgca 1200
tttattttct tagtgacaag ggtgataaac tcaaatacag cttttagaac tggttacaat 1260
agcgacggag agttaggtta ttgggataag ttagagccac tttatacaat ttttgatggt 1320
gtatctaaaa cattctctgg tatttggact cctgtaaaga atgacttcaa agagttttat 1380
gatttatacc tttctgatgt agagaaatat aatggttcgg ggaaattgtt tcccaaaaca 1440
cctatacctg aaaatgcttt ttctctttct attattccat ggacttcatt tactgggttt 1500
aacttaaata tcaataataa tagtaattac cttctaccca ttattacagc aggaaaattc 1560
attaataaag gtaattcaat atatttaccg ctatctttac aggtacatca ttctgtttgt 1620
gatggttatc atgcaggatt gtttatgaac tctattcagg aattgtcaga taggcctaat 1680
gactggcttt tataatatga gataatgccg actgtacttt ttacagtcgg ttttctaatg 1740
tcactaacct gccccgttag ttgaagaagg tttttatatt acagctccag atccatatcc 1800
ttctttttct gaaccgactt ctcctttttc gcttctttat tccaattgct ttattgacgt 1860
tgagcctcgg aacccttaac aatcccaaaa cttgtcgaat ggtcggctta atagctcacg 1920
ctatgccgac attcgtctgc aagtttagtt aagggttctt ctcaacgcac aataaatttt 1980
ctcggcataa atgcgtggtc taatttttat ttttaataac cttgatagca aaaaatgcca 2040
ttccaataca aaaccacata cctataatcg ataaccacat aacagtcata aaaccactcc 2100
tttttaacaa actttatcac aagaaatatt tacccggggt ccagaaggtc gatagaaagc 2160
gtgagaaaca gcgtacagac gatttagaga tgtagaggta cttttatgcc gagaaaactt 2220
tttgcgtgtg acagtcctta aaatatactt agagcgtaag cgaaagtagt agcgacagct 2280
attaactttc ggttgcaaag ctctaggatt tttaatggac gcagcgcatc acacgcaaaa 2340
aggaaattgg aataaatgcg aaatttgaga tgttaattaa agaccttttt gaggtctttt 2400
tttcttagat ttttggggtt atttagggga gaaaacatag gggggtacta cgacctcccc 2460
cctaggtgtc cattgtccat tgtccaaaca aataaataaa tattgggttt ttaatgttaa 2520
aaggttgttt tttatgttaa agtgaaaaaa acagatgttg ggaggtacag tgatggttgt 2580
agatagaaaa gaagagaaaa aagttgctgt tactttaaga cttacaacag aagaaaatga 2640
gatattaaat agaatcaaag aaaaatataa tattagcaaa tcagatgcaa ccggtattct 2700
aataaaaaaa tatgcaaagg aggaatacgg tgcattttaa acaaaaaaag atagacagca 2760
ctggcatgct gcctatctat gactaaattt tgttaagtgt attagcaccg ttattatatc 2820
atgagcgaaa atgtaataaa agaaactgaa aacaagaaaa attcaagagg acgtaattgg 2880
acatttgttt tatatccaga atcagcaaaa gccgagtggt tagagtattt aaaagagtta 2940
cacattcaat ttgtagtgtc tccattacat gatagggata ctgatacaga aggtaggatg 3000
aaaaaagagc attatcatat tctagtgatg tatgagggta ataaatctta tgaacagata 3060
aaaataatta acagaagaat tgaatgcgac tattccgcag attgcaggaa gtgtgaaagg 3120
tcttgtgaga tatatgcttc acatggacga tcctaataaa tttaaatatc aaaaagaaga 3180
tatgatagtt tatggcggtg tagatgttga tgaattatta aagaaaacaa caacagatag 3240
atataaatta attaaagaaa tgattgagtt tattgatgaa caaggaatcg tagaatttaa 3300
gagtttaatg gattatgcaa tgaagtttaa atttgatgat tggttcccgc ttttatgtga 3360
taactcggcg tatgttattc aagaatatat aaaatcaaat cggtataaat ctgaccgata 3420
gattttgaat ttaggtgtca caagacactc ttttttcgca ccagcgaaaa ctggtttaag 3480
ccgactgcgc aaaagacata atcgattcac aaaaaatagg cacacgaaaa acaagttaag 3540
ggatgcagtt tatgcatccc ttaacggtac cactggccgt cgttttacaa cgtcgtgact 3600
gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 3660
ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 3720
gcgaatggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 3780
tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc 3840
cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 3900
aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 3960
gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa 4020
tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 4080
tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 4140
ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 4200
ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 4260
aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 4320
gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 4380
ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 4440
gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 4500
cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 4560
cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 4620
acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 4680
caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 4740
taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 4800
ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 4860
aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 4920
agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 4980
atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 5040
tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 5100
tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 5160
gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 5220
taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 5280
aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 5340
ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 5400
catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 5460
ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 5520
ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 5580
agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 5640
taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 5700
atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 5760
cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 5820
ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 5880
accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 5940
gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc 6000
gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg 6060
agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta 6120
tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca 6180
gctatgacca tgattacgcc aagcttgcat gcccgcgggg atccgctagc tctaga 6236
Claims (6)
1.一种高产表面活性素的基因工程菌,其特征在于,所述基因工程菌以解淀粉芽孢杆菌为出发菌株,敲除或者失活菌株基因组中ComQ基因所得;所述ComQ基因的核苷酸序列如SEQ ID NO.1所示;所述解淀粉芽孢杆菌为解淀粉芽孢杆菌CPLK1314,保藏号CCTCC NO:M2017658。
2.一种权利要求1所述高产表面活性素的基因工程菌的构建方法,其特征在于,包括如下步骤:
(1)以ComQ基因核酸序列设计引物,以解淀粉芽孢杆菌的基因组DNA作为模板,扩增到ComQ基因片段;
(2)同源重组质粒载体pTCCOMQ的构建:扩增的ComQ基因片段经过XhoⅠ和EcoRⅠ双酶切,然后经过T4DNA连接酶连接到经过同样双酶切的质粒载体pTC中,构建同源重组整合质粒载体pTCCOMQ;
(3)ComQ基因失活突变菌株ΔComQ的构建:将构建好的重组质粒pTCCOMQ转化至解淀粉芽孢杆菌得到高产表面活性素的基因工程菌ComQ基因突变株ΔComQ。
3.根据权利要求2的构建方法,其特征在于,步骤(1)所述的引物为: COMQF 5′-TTTGAATTCGGGATTCATATAGCTAAGAA-3′和COMQ-R 5′- TTTCTCGAGAAATAAATCATTTTTTAATTG -3′。
4.一种权利要求1所述高产表面活性素的基因工程菌在发酵生产表面活性素中的应用。
5.根据权利要求4所述的应用,其特征在于,所述发酵为通过基因工程菌进行发酵罐发酵:将菌种活化后制备种子液,按照体积比10-20%接种量接入新鲜的发酵培养基,开始发酵,发酵过程中控制pH稳定在6.5左右,温度维持在30℃,溶氧在25-35%之间;发酵周期40-50h。
6.根据权利要求5所述的应用,其特征在于,所述发酵培养基组成为:葡萄糖10-15g/L,蔗糖 10-15g/L,酵母粉5-8g/L, 谷氨酸钠5-6g/ L,蛋白胨1-5g/L,K2HPO4 1g/L,KH2PO41g/L,MgSO4·7H2O 500 mg/L,FeSO4·7H2O 10mg/L,MnSO4·H2O 10mg/L,VB1、VB3、VB5、VB12、VH各1-3mg/L,其余为水,pH 7.0。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011472978.XA CN112625986B (zh) | 2020-12-15 | 2020-12-15 | 一种高产表面活性素的基因工程菌及其构建方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011472978.XA CN112625986B (zh) | 2020-12-15 | 2020-12-15 | 一种高产表面活性素的基因工程菌及其构建方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112625986A CN112625986A (zh) | 2021-04-09 |
CN112625986B true CN112625986B (zh) | 2023-01-31 |
Family
ID=75312790
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011472978.XA Active CN112625986B (zh) | 2020-12-15 | 2020-12-15 | 一种高产表面活性素的基因工程菌及其构建方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112625986B (zh) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113980991B (zh) * | 2021-11-04 | 2024-03-26 | 淮阴工学院 | 大肠杆菌-芽孢杆菌穿梭质粒载体及其构建方法和应用 |
CN114292867B (zh) * | 2021-12-31 | 2024-01-23 | 淮阴工学院 | 芽孢杆菌表达载体及其构建方法和应用 |
CN114292864B (zh) * | 2021-12-31 | 2023-08-08 | 淮阴工学院 | 高产表面活性素的贝莱斯芽孢杆菌突变株及其构建方法和应用 |
CN114806986B (zh) * | 2022-03-29 | 2023-11-14 | 淮阴工学院 | 高产罗克霉素的基因工程菌及其构建方法和应用 |
CN115308342A (zh) * | 2022-10-11 | 2022-11-08 | 海南康植肽生物科技有限公司 | 一种表面活性素的检测方法 |
-
2020
- 2020-12-15 CN CN202011472978.XA patent/CN112625986B/zh active Active
Also Published As
Publication number | Publication date |
---|---|
CN112625986A (zh) | 2021-04-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109797111B (zh) | 一种产苹果酸基因工程菌及其生产苹果酸的方法 | |
CN112625986B (zh) | 一种高产表面活性素的基因工程菌及其构建方法和应用 | |
CN100540667C (zh) | 利用水稻胚乳细胞作为生物反应器生产重组人血清白蛋白 | |
KR101258949B1 (ko) | 활성형 재조합 혈액응고 9인자의 대량생산 방법 | |
CN112877351A (zh) | 一种用于防治新冠病毒感染的重组质粒、重组乳酸杆菌表达系统及其应用 | |
CN110438053B (zh) | 一种适用于聚球藻的生物封存系统、构建方法及应用 | |
CN113980991B (zh) | 大肠杆菌-芽孢杆菌穿梭质粒载体及其构建方法和应用 | |
CN108884446A (zh) | 基于aav的条件表达系统 | |
CN111269869B (zh) | 一种重组丙酮丁醇梭菌的构建方法以及在发酵半纤维制备丁醇中的应用 | |
CN114107390B (zh) | 一种用于表达抗体IgG1的rAAV载体及其应用 | |
CN113308482B (zh) | 云南腾冲来源四氢嘧啶合成基因簇及其应用 | |
CN112626064B (zh) | 一种新型转座子突变株文库的构建系统 | |
CN110184292B (zh) | 一种利用分子伴侣提高酵母细胞表面展示功能性Infliximab Fab片段的方法 | |
CN110938648B (zh) | 一种真菌分泌表达载体、构建方法及其应用 | |
CN109872774B (zh) | 一种基于yess分析原核生物中蛋白相互作用的方法 | |
CN109735558B (zh) | 一种重组car19-il24基因、慢病毒载体、car19-il24-t细胞及应用 | |
CN111088204A (zh) | 表达Caspase-3重组scFv78的重组大肠杆菌及其功能验证方法 | |
CN115161294B (zh) | 新城疫疫苗株及其构建方法、禽类免疫识别方法、应用 | |
CN112180087B (zh) | 检测鸭疫里默氏杆菌抗体的elisa方法及其试剂盒和应用 | |
CN114807194B (zh) | 一种提高梭菌中蛋白表达效率的方法 | |
CN110679606B (zh) | dsRNA及其在防治埃及伊蚊中的应用 | |
CN107142259A (zh) | 一种表达外源基因的启动子及其应用 | |
CN111909850B (zh) | 基于杜氏盐藻代谢途径和夏侧金盏花cbfd与hbfd的产虾青素工程菌及其构建与应用 | |
CN111206044A (zh) | 一种新的质粒载体构建方法 | |
CN111117973A (zh) | 一种受microRNA调控的重组溶瘤肠道病毒71型及应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20210409 Assignee: Nanjing Baiskai Technology Co.,Ltd. Assignor: HUAIYIN INSTITUTE OF TECHNOLOGY Contract record no.: X2023980041333 Denomination of invention: A Genetically Engineered Strain with High Yield of Surfactants and Its Construction Method and Application Granted publication date: 20230131 License type: Common License Record date: 20230907 |
|
EE01 | Entry into force of recordation of patent licensing contract |