CN108865918A - A method of the gram-positive bacterium of label survival and its cell wall - Google Patents
A method of the gram-positive bacterium of label survival and its cell wall Download PDFInfo
- Publication number
- CN108865918A CN108865918A CN201710321663.7A CN201710321663A CN108865918A CN 108865918 A CN108865918 A CN 108865918A CN 201710321663 A CN201710321663 A CN 201710321663A CN 108865918 A CN108865918 A CN 108865918A
- Authority
- CN
- China
- Prior art keywords
- bacterium
- gram
- acetyl
- glucosamine
- detectable marker
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000894006 Bacteria Species 0.000 title claims abstract description 111
- 238000000034 method Methods 0.000 title claims abstract description 55
- 210000002421 cell wall Anatomy 0.000 title claims abstract description 43
- 230000004083 survival effect Effects 0.000 title claims abstract description 25
- 239000003550 marker Substances 0.000 claims abstract description 80
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 47
- 150000004676 glycans Chemical class 0.000 claims abstract description 43
- 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 claims abstract description 38
- 229950006780 n-acetylglucosamine Drugs 0.000 claims abstract description 37
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 claims abstract description 36
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 claims abstract description 36
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 35
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 33
- TZRXHJWUDPFEEY-UHFFFAOYSA-N Pentaerythritol Tetranitrate Chemical class [O-][N+](=O)OCC(CO[N+]([O-])=O)(CO[N+]([O-])=O)CO[N+]([O-])=O TZRXHJWUDPFEEY-UHFFFAOYSA-N 0.000 claims abstract description 31
- 238000006243 chemical reaction Methods 0.000 claims abstract description 26
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 claims description 73
- 125000000304 alkynyl group Chemical group 0.000 claims description 36
- 229910000073 phosphorus hydride Inorganic materials 0.000 claims description 31
- 230000001580 bacterial effect Effects 0.000 claims description 26
- 238000012986 modification Methods 0.000 claims description 24
- 230000004048 modification Effects 0.000 claims description 24
- 239000001963 growth medium Substances 0.000 claims description 14
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 claims description 13
- 239000013049 sediment Substances 0.000 claims description 8
- 241000192125 Firmicutes Species 0.000 claims description 4
- 239000010985 leather Substances 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 4
- 210000004027 cell Anatomy 0.000 abstract description 15
- 230000037353 metabolic pathway Effects 0.000 abstract description 4
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 abstract description 2
- 108010013639 Peptidoglycan Proteins 0.000 abstract 1
- 238000010348 incorporation Methods 0.000 abstract 1
- 150000001875 compounds Chemical class 0.000 description 34
- 229910052757 nitrogen Inorganic materials 0.000 description 25
- 239000000126 substance Substances 0.000 description 25
- 102000004190 Enzymes Human genes 0.000 description 23
- 108090000790 Enzymes Proteins 0.000 description 23
- 229940088598 enzyme Drugs 0.000 description 23
- -1 acyl aminoglucose Chemical compound 0.000 description 20
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 17
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 17
- 230000001376 precipitating effect Effects 0.000 description 13
- 239000000872 buffer Substances 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- 229910019142 PO4 Inorganic materials 0.000 description 9
- 239000007853 buffer solution Substances 0.000 description 9
- 239000000975 dye Substances 0.000 description 9
- 150000002772 monosaccharides Chemical class 0.000 description 9
- 239000012071 phase Substances 0.000 description 9
- 239000010452 phosphate Substances 0.000 description 9
- 230000003595 spectral effect Effects 0.000 description 9
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 8
- 235000002639 sodium chloride Nutrition 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 239000002253 acid Substances 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 7
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- 102000016938 Catalase Human genes 0.000 description 6
- 108010053835 Catalase Proteins 0.000 description 6
- 101710088194 Dehydrogenase Proteins 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 6
- 150000001345 alkine derivatives Chemical class 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 229910000397 disodium phosphate Inorganic materials 0.000 description 6
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 6
- 125000001820 oxy group Chemical group [*:1]O[*:2] 0.000 description 6
- 125000001476 phosphono group Chemical group [H]OP(*)(=O)O[H] 0.000 description 6
- 230000002285 radioactive effect Effects 0.000 description 6
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 6
- 229910000162 sodium phosphate Inorganic materials 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- LCGTWRLJTMHIQZ-UHFFFAOYSA-N 5H-dibenzo[b,f]azepine Chemical compound C1=CC2=CC=CC=C2NC2=CC=CC=C21 LCGTWRLJTMHIQZ-UHFFFAOYSA-N 0.000 description 5
- 241000193830 Bacillus <bacterium> Species 0.000 description 5
- 239000013522 chelant Substances 0.000 description 5
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- WQZIDRAQTRIQDX-UHFFFAOYSA-N 6-carboxy-x-rhodamine Chemical compound OC(=O)C1=CC=C(C([O-])=O)C=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 WQZIDRAQTRIQDX-UHFFFAOYSA-N 0.000 description 4
- VWOLRKMFAJUZGM-UHFFFAOYSA-N 6-carboxyrhodamine 6G Chemical compound [Cl-].C=12C=C(C)C(NCC)=CC2=[O+]C=2C=C(NCC)C(C)=CC=2C=1C1=CC(C(O)=O)=CC=C1C(=O)OCC VWOLRKMFAJUZGM-UHFFFAOYSA-N 0.000 description 4
- CJIJXIFQYOPWTF-UHFFFAOYSA-N 7-hydroxycoumarin Natural products O1C(=O)C=CC2=CC(O)=CC=C21 CJIJXIFQYOPWTF-UHFFFAOYSA-N 0.000 description 4
- 108010022752 Acetylcholinesterase Proteins 0.000 description 4
- 102100033639 Acetylcholinesterase Human genes 0.000 description 4
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 4
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 4
- 108090001008 Avidin Proteins 0.000 description 4
- KWDRYXUNQUYJCF-UHFFFAOYSA-N ClCCCl.C12CC3CC(CC(C1)C3)C2 Chemical compound ClCCCl.C12CC3CC(CC(C1)C3)C2 KWDRYXUNQUYJCF-UHFFFAOYSA-N 0.000 description 4
- PQMOXTJVIYEOQL-UHFFFAOYSA-N Cumarin Natural products CC(C)=CCC1=C(O)C(C(=O)C(C)CC)=C(O)C2=C1OC(=O)C=C2CCC PQMOXTJVIYEOQL-UHFFFAOYSA-N 0.000 description 4
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 4
- 102100022624 Glucoamylase Human genes 0.000 description 4
- 108010015776 Glucose oxidase Proteins 0.000 description 4
- 239000004366 Glucose oxidase Substances 0.000 description 4
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 102000013460 Malate Dehydrogenase Human genes 0.000 description 4
- 108010026217 Malate Dehydrogenase Proteins 0.000 description 4
- FSOGIJPGPZWNGO-UHFFFAOYSA-N Meomammein Natural products CCC(C)C(=O)C1=C(O)C(CC=C(C)C)=C(O)C2=C1OC(=O)C=C2CCC FSOGIJPGPZWNGO-UHFFFAOYSA-N 0.000 description 4
- 108010059724 Micrococcal Nuclease Proteins 0.000 description 4
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 108010052090 Renilla Luciferases Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 102000005924 Triose-Phosphate Isomerase Human genes 0.000 description 4
- 108700015934 Triose-phosphate isomerases Proteins 0.000 description 4
- 229940022698 acetylcholinesterase Drugs 0.000 description 4
- AORNIVFSXFONEC-UHFFFAOYSA-N acridine-1-carboxamide Chemical compound C1=CC=C2C=C3C(C(=O)N)=CC=CC3=NC2=C1 AORNIVFSXFONEC-UHFFFAOYSA-N 0.000 description 4
- 102000005936 beta-Galactosidase Human genes 0.000 description 4
- 108010005774 beta-Galactosidase Proteins 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 4
- 229940116332 glucose oxidase Drugs 0.000 description 4
- 235000019420 glucose oxidase Nutrition 0.000 description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 4
- 150000002460 imidazoles Chemical class 0.000 description 4
- KNJDBYZZKAZQNG-UHFFFAOYSA-N lucigenin Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.C12=CC=CC=C2[N+](C)=C(C=CC=C2)C2=C1C1=C(C=CC=C2)C2=[N+](C)C2=CC=CC=C12 KNJDBYZZKAZQNG-UHFFFAOYSA-N 0.000 description 4
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 description 4
- 239000004033 plastic Substances 0.000 description 4
- 229920003023 plastic Polymers 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- MYIOYATURDILJN-UHFFFAOYSA-N rhodamine 110 Chemical compound [Cl-].C=12C=CC(N)=CC2=[O+]C2=CC(N)=CC=C2C=1C1=CC=CC=C1C(O)=O MYIOYATURDILJN-UHFFFAOYSA-N 0.000 description 4
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 239000005315 stained glass Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 201000008827 tuberculosis Diseases 0.000 description 4
- HFTAFOQKODTIJY-UHFFFAOYSA-N umbelliferone Natural products Cc1cc2C=CC(=O)Oc2cc1OCC=CC(C)(C)O HFTAFOQKODTIJY-UHFFFAOYSA-N 0.000 description 4
- ORHBXUUXSCNDEV-UHFFFAOYSA-N umbelliferone Chemical compound C1=CC(=O)OC2=CC(O)=CC=C21 ORHBXUUXSCNDEV-UHFFFAOYSA-N 0.000 description 4
- QGKMIGUHVLGJBR-UHFFFAOYSA-M (4z)-1-(3-methylbutyl)-4-[[1-(3-methylbutyl)quinolin-1-ium-4-yl]methylidene]quinoline;iodide Chemical compound [I-].C12=CC=CC=C2N(CCC(C)C)C=CC1=CC1=CC=[N+](CCC(C)C)C2=CC=CC=C12 QGKMIGUHVLGJBR-UHFFFAOYSA-M 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 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108010024976 Asparaginase Proteins 0.000 description 3
- 102000015790 Asparaginase Human genes 0.000 description 3
- 241000193738 Bacillus anthracis Species 0.000 description 3
- XQSPNYNSJBNQHQ-DBKUKYHUSA-N C(C)(=O)C1(O)[C@H](N)[C@@H](O)[C@H](O)[C@H](O1)CO.[N] Chemical compound C(C)(=O)C1(O)[C@H](N)[C@@H](O)[C@H](O)[C@H](O1)CO.[N] XQSPNYNSJBNQHQ-DBKUKYHUSA-N 0.000 description 3
- 241000193449 Clostridium tetani Species 0.000 description 3
- 241000186216 Corynebacterium Species 0.000 description 3
- 241000186227 Corynebacterium diphtheriae Species 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 241000194033 Enterococcus Species 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 241000186781 Listeria Species 0.000 description 3
- 102000016943 Muramidase Human genes 0.000 description 3
- 108010014251 Muramidase Proteins 0.000 description 3
- 241000186359 Mycobacterium Species 0.000 description 3
- NMOJAXCSURVGEY-UHFFFAOYSA-N N#CC#N.[S] Chemical compound N#CC#N.[S] NMOJAXCSURVGEY-UHFFFAOYSA-N 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 3
- 241000191940 Staphylococcus Species 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 108010090804 Streptavidin Proteins 0.000 description 3
- 241000194017 Streptococcus Species 0.000 description 3
- 241000193998 Streptococcus pneumoniae Species 0.000 description 3
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 229960003272 asparaginase Drugs 0.000 description 3
- DCXYFEDJOCDNAF-UHFFFAOYSA-M asparaginate Chemical compound [O-]C(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-M 0.000 description 3
- 229940065181 bacillus anthracis Drugs 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N biotin Natural products N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 235000009508 confectionery Nutrition 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000003818 flash chromatography Methods 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 229960000274 lysozyme Drugs 0.000 description 3
- 239000004325 lysozyme Substances 0.000 description 3
- 235000010335 lysozyme Nutrition 0.000 description 3
- 230000002503 metabolic effect Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 229910052707 ruthenium Inorganic materials 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- VGNCBRNRHXEODV-XXVHXNRLSA-N (6r,7r)-1-[(4s,5r)-4-acetyloxy-5-methyl-3-methylidene-6-phenylhexyl]-6-dodecoxy-4,7-dihydroxy-2,8-dioxabicyclo[3.2.1]octane-3,4,5-tricarboxylic acid Chemical compound C([C@@H](C)[C@H](OC(C)=O)C(=C)CCC12[C@H](O)[C@H](C(O2)(C(O)=O)C(O)(C(O1)C(O)=O)C(O)=O)OCCCCCCCCCCCC)C1=CC=CC=C1 VGNCBRNRHXEODV-XXVHXNRLSA-N 0.000 description 2
- QRZUPJILJVGUFF-UHFFFAOYSA-N 2,8-dibenzylcyclooctan-1-one Chemical compound C1CCCCC(CC=2C=CC=CC=2)C(=O)C1CC1=CC=CC=C1 QRZUPJILJVGUFF-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- ZUHQCDZJPTXVCU-UHFFFAOYSA-N C1#CCCC2=CC=CC=C2C2=CC=CC=C21 Chemical compound C1#CCCC2=CC=CC=C2C2=CC=CC=C21 ZUHQCDZJPTXVCU-UHFFFAOYSA-N 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 241000605909 Fusobacterium Species 0.000 description 2
- 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 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
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- DUKURNFHYQXCJG-UHFFFAOYSA-N Lewis A pentasaccharide Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(C(O)C(O)C(CO)O2)O)C(NC(C)=O)C(OC2C(C(OC3C(OC(O)C(O)C3O)CO)OC(CO)C2O)O)OC1CO DUKURNFHYQXCJG-UHFFFAOYSA-N 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 description 2
- 201000005010 Streptococcus pneumonia Diseases 0.000 description 2
- AIGAZQPHXLWMOJ-UHFFFAOYSA-N Tanshinone I Chemical compound C1=CC2=C(C)C=CC=C2C(C(=O)C2=O)=C1C1=C2C(C)=CO1 AIGAZQPHXLWMOJ-UHFFFAOYSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 2
- 238000006352 cycloaddition reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- ZRSNZINYAWTAHE-UHFFFAOYSA-N p-methoxybenzaldehyde Chemical compound COC1=CC=C(C=O)C=C1 ZRSNZINYAWTAHE-UHFFFAOYSA-N 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- YGOAYNVRTAEAAI-GKZUZGRISA-N (2R)-2-[(3R,4R,5S,6R)-2-acetyl-3-amino-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxypropanoic acid Chemical compound C(C)(=O)C1(O)[C@H](N)[C@@H](O[C@@H](C(=O)O)C)[C@H](O)[C@H](O1)CO YGOAYNVRTAEAAI-GKZUZGRISA-N 0.000 description 1
- JCZPMGDSEAFWDY-SQOUGZDYSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanamide Chemical compound NC(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO JCZPMGDSEAFWDY-SQOUGZDYSA-N 0.000 description 1
- FOLCUFKJHSQMEL-BIXPGCQOSA-N (4-butylcyclohexyl) N-[(2S)-4-methyl-1-oxo-1-[[(2S)-1-oxo-3-[(3S)-2-oxopyrrolidin-3-yl]propan-2-yl]amino]pentan-2-yl]carbamate Chemical compound CCCCC1CCC(CC1)OC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C[C@@H]2CCNC2=O)C=O FOLCUFKJHSQMEL-BIXPGCQOSA-N 0.000 description 1
- SJEYUYPBCPRFNM-WQWHYOKSSA-N (6S)-4-[(2E,4E)-hexa-2,4-dienoyl]-3,6-dihydroxy-2,6-dimethylcyclohexa-2,4-dien-1-one Chemical compound C\C=C\C=C\C(=O)C1=C[C@](C)(O)C(=O)C(C)=C1O SJEYUYPBCPRFNM-WQWHYOKSSA-N 0.000 description 1
- UXKLQDCALAWFIU-VKNDCNMPSA-N (6r,7r)-1-[(4s,5r)-4-acetyloxy-5-methyl-3-methylidene-6-phenylhexyl]-4,7-dihydroxy-6-tetradecoxy-2,8-dioxabicyclo[3.2.1]octane-3,4,5-tricarboxylic acid Chemical compound C([C@@H](C)[C@H](OC(C)=O)C(=C)CCC12[C@H](O)[C@H](C(O2)(C(O)=O)C(O)(C(O1)C(O)=O)C(O)=O)OCCCCCCCCCCCCCC)C1=CC=CC=C1 UXKLQDCALAWFIU-VKNDCNMPSA-N 0.000 description 1
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 238000007115 1,4-cycloaddition reaction Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- MSFSPUZXLOGKHJ-PGYHGBPZSA-N 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucopyranose Chemical compound OC(=O)[C@@H](C)O[C@@H]1[C@@H](N)C(O)O[C@H](CO)[C@H]1O MSFSPUZXLOGKHJ-PGYHGBPZSA-N 0.000 description 1
- BZSVVCFHMVMYCR-UHFFFAOYSA-N 2-pyridin-2-ylpyridine;ruthenium Chemical compound [Ru].N1=CC=CC=C1C1=CC=CC=N1.N1=CC=CC=C1C1=CC=CC=N1.N1=CC=CC=C1C1=CC=CC=N1 BZSVVCFHMVMYCR-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 241000432824 Asparagus densiflorus Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 206010013786 Dry skin Diseases 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- LSDPWZHWYPCBBB-UHFFFAOYSA-N Methanethiol Chemical compound SC LSDPWZHWYPCBBB-UHFFFAOYSA-N 0.000 description 1
- 241000186366 Mycobacterium bovis Species 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical class [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 238000003800 Staudinger reaction Methods 0.000 description 1
- 229930183118 Tanshinone Natural products 0.000 description 1
- KDWANXLZQZVUMF-LZQZFOIKSA-N [(2R,3R,4R,5R,6R)-5-acetamido-4-acetyloxy-2-(azidomethyl)-6-phosphonooxyoxan-3-yl] acetate Chemical compound C(C)(=O)N[C@@H]1[C@H]([C@@H]([C@H](O[C@@H]1OP(=O)(O)O)CN=[N+]=[N-])OC(C)=O)OC(C)=O KDWANXLZQZVUMF-LZQZFOIKSA-N 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000010461 azide-alkyne cycloaddition reaction Methods 0.000 description 1
- 230000037358 bacterial metabolism Effects 0.000 description 1
- CSKNSYBAZOQPLR-UHFFFAOYSA-N benzenesulfonyl chloride Chemical compound ClS(=O)(=O)C1=CC=CC=C1 CSKNSYBAZOQPLR-UHFFFAOYSA-N 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 150000001721 carbon Chemical group 0.000 description 1
- 239000002041 carbon nanotube Substances 0.000 description 1
- 229910021393 carbon nanotube Inorganic materials 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000012916 chromogenic reagent Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- LNHSQAOQVNHUGL-QRBHCBQLSA-N dbco-peg4-biotin Chemical compound C1C2=CC=CC=C2C#CC2=CC=CC=C2N1C(=O)CCNC(=O)CCOCCOCCOCCOCCNC(=O)CCCC[C@H]1[C@H]2NC(=O)N[C@H]2CS1 LNHSQAOQVNHUGL-QRBHCBQLSA-N 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- 239000000412 dendrimer Substances 0.000 description 1
- 229920000736 dendritic polymer Polymers 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000012407 engineering method Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000037347 glycan metabolism Effects 0.000 description 1
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000002075 main ingredient Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 108010009719 mutanolysin Proteins 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000004260 plant-type cell wall biogenesis Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-O pyridinium Chemical compound C1=CC=[NH+]C=C1 JUJWROOIHBZHMG-UHFFFAOYSA-O 0.000 description 1
- 239000000941 radioactive substance Substances 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- SJEYUYPBCPRFNM-UHFFFAOYSA-N sorbicillinol Natural products CC=CC=CC(=O)C1=CC(C)(O)C(=O)C(=C1O)C SJEYUYPBCPRFNM-UHFFFAOYSA-N 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 125000003698 tetramethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 1
- JGVWCANSWKRBCS-UHFFFAOYSA-N tetramethylrhodamine thiocyanate Chemical compound [Cl-].C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=C(SC#N)C=C1C(O)=O JGVWCANSWKRBCS-UHFFFAOYSA-N 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- AVBGNFCMKJOFIN-UHFFFAOYSA-N triethylammonium acetate Chemical compound CC(O)=O.CCN(CC)CC AVBGNFCMKJOFIN-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the methods that a kind of cell wall of gram-positive bacterium to survival is marked.The method passes through bio-orthogonal reaction; in the full acetylated N-acetyl-glucosamine -1- phosphoric acid incorporation whole cell peptidoglycan for being modified nitrine using the metabolic pathway of bacterium itself; it is then reacted by click chemistry, detectable marker is coupled to peptide glycan, to realize the label to bacterium peptide glycan.The method has universality, can be used for quickly and conveniently marking gram-positive bacterium.
Description
Technical field
The present invention relates to one kind by bio-orthogonal reaction, and the leather of survival is marked using the biosynthesis pathway of cell wall
The method of gram-positive bacteria.
Background technique
It is currently known the cell surface marker technology of various bacteria.For example, can be used expression can be with bacterial surface protein
In conjunction with target protein gene engineering method.However, such cellular degeneration necrosis method is only applicable to labelled protein and more
Peptides.Compared with genetic manipulation method, the chemical method based on metabolic pathway can be utilized by adding metabolic analogue small molecule
Latter functionalities label is covalently attached to different non-natural epitopes by the metabolic pathway of cell itself.
One of typical composition of gram positive bacterial cell wall is peptide glycan.Peptide glycan is a kind of network-like miscellaneous more
Sugar, skeleton are made of peptide and glycan two parts, and peptide therein includes bridge two parts between tetrapeptide tail and peptide, and glycan is by N- second
The long-chain that two kinds of monosaccharide of acyl aminoglucose (G) and -acetylmuramic acid (M) are alternately connected to by β -1,4 glycosidic bond.Eukaryocyte
In be free of peptide glycan, therefore, peptide glycan is good inhibits or the target of label bacterium.However currently, to peptide glycan in bacterium
In biosynthesis pathway know little about it.The research of peptide glycan is also only limitted to find it as the potential new of inhibitor target
Purposes.Nearly ten years, the research for the synthesis of peptide glycan being tracked or being edited makes great progress.The peptide reported recently
The imaging method of glycan including the use of fluorescent marker lectin (or antibody), using can act on cell wall with fluorescence
The antibiotic of label, the strategy based on the metabolic marker for carrying out detection or radioactive substance tracer to free mercaptan, using glimmering
The enzyme process etc. of light substrate.Although these technologies have been used to disclose the dynamic process of bacteria cell wall peptide glycan metabolism, however,
These technical operations are excessively many and diverse and resolution ratio is lower, and are only applicable to specific bacterium kind, in terms of the universality of application
There are significant limitations.In the above-described techniques, the antibody technique of fluorescent marker is because its is easy to operate, substance is molten in a short time
Solution property is higher and most widely used.However, the antibody of fluorescent marker can inhibit the activity of bacterium, it is difficult at the beginning of its labeling and growing
The gram-positive bacteria of phase.There are still the demands for not interfering the peptide glycan labeling method of bacterial growth at present.
Bio-orthogonal reaction refers to chemical reaction with the following characteristics：It carries out in physiological conditions, does not interfere while sending out
Raw other biochemical reactions or various biological endogenous processes, and organism and biological endogenous molecule will not be caused to damage.At present
Most common bio-orthogonal reaction group is to the azido-including click chemistry (Click Chemistry) reaction can occur
Alkynyl and azido-triaryl phosphine.Wherein, the staudinger reaction (Staudinger that azido and triaryl phosphine occur
A member with the bio-orthogonal reaction group centering can be connected by the cycloaddition reaction Ligation) or with alkynyl occurred
Sugar be coupled to the detectable marker being connected with another member of the bio-orthogonal reaction group centering, realize detectable mark
Note object is modified to the purpose of sugar.Wherein, the cycloaddition reaction (Strain-promoted of azido group and high-tension alkynes
Azide-alkyne cycloaddition, SPAAC) because it does not need to become research due to adding catalyst and applies hot spot.From
Since click chemistry reaction in 2001 is suggested, which has been widely used in surface modification, dendrimer and function
Generation, cell marking, biological medicine synthesis, nucleic acid or modification of carbon nanotube of polymer etc..CN104039977B is proposed
A method of gram-negative bacterial cell wall lipopolysaccharides is marked using azido-alkynyl bio-orthogonal reaction.However, at present
The method that the cell wall of gram-positive bacterium is marked is not reported also.
Summary of the invention
The present invention is directed to the metabolic pathways using gram-positive bacterium itself, and metabolic analogue small molecule is added to carefully
Cell wall, and the method that the gram-positive bacterium of survival and its cell wall are marked is developed based on bio-orthogonal reaction.
On the one hand, the purpose of the present invention is to provide a kind of method that the gram-positive bacterium to survival is marked,
Described method includes following steps：
(i) the full acetylated N-acetyl-glucosamine -1- phosphoric acid of nitrine modification is added in gram-positive bacterium culture medium,
Gram-positive bacterium is inoculated in the culture medium and the gram-positive bacterium is cultivated, obtains Bacteria Culture
Object；
(ii) bacterial cultures that collection step (i) obtains obtains the precipitating of the bacterial cultures by processing
Object；
(iii) bacterium in the sediment obtained to step (ii) is fixed；
(iv) to the detectable label for being connected with alkynyl or triaryl phosphine through adding in fixed bacterium of step (iii)
Object, so that the marker is coupled to the peptide glycan in the bacterium；
(v) detectable marker is detected.
On the other hand, the present invention provides the full acetylated N-acetyl-glucosamine -1- phosphoric acid of nitrine modification to be used for survival
Gram-positive bacterium the purposes that is marked of cell wall, wherein the full acetylated N- acetyl modified by the nitrine
The bio-orthogonal reaction of the azido of aminoglucose -1- phosphoric acid and the alkynyl or triaryl phosphine that are connected to detectable marker is realized
To the label of the cell wall of the gram-positive bacterium of survival.
Another aspect, the present invention provides nitrine modification full acetylated N-acetyl-glucosamine -1- phosphoric acid be used for comprising
The purposes of the gram-positive bacterium of survival is specifically marked in the sample of bacterium, wherein modify by the nitrine complete
The azido of acetylation N-acetyl-glucosamine -1- phosphoric acid be connected to detectable marker alkynyl or triaryl phosphine biology just
Reaction is handed over to realize the label of the cell wall to the gram-positive bacterium of survival.
In another aspect, the present invention provides a kind of for detecting the kit of the gram-positive bacterium of survival, wherein institute
State full acetylated N-acetyl-glucosamine -1- phosphoric acid that kit includes nitrine modification and be connected with alkynyl or triaryl phosphine can
Detect marker.
Detailed description of the invention
Fig. 1 is according to embodiment 2, the HPLC map of experimental group peptide glycan.
Fig. 2 is 6- azido methyl-N-acetyl-glucosamine LC-MS map in experimental group according to embodiment 2.
Fig. 3 is the LC-MS map of N-acetyl-glucosamine in experimental group according to embodiment 2.
Fig. 4 is according to embodiment 2, the HPLC map of control group peptide glycan.
Fig. 5 is according to embodiment 2, the LC-MS map of control group monosaccharide (N-acetyl-glucosamine).
Fig. 6 is according to embodiment 3, the fluorescence results figure (100 times of amplification factor) of experimental group BCG bacterium cell wall label.Its
In, 1 is light field, and 2 be the channel FITC, and 3 be the channel TXRED, and 4 be 1,2,3 stacking chart.
Fig. 7 is the enlarged drawing of the single bacterium in Fig. 6.Wherein, 1 is light field, and 2 be the channel FITC, and 3 be the channel TXRED, 4
For 1,2,3 stacking chart.
Fig. 8 is according to embodiment 3, the fluorescence results figure (100 times of amplification factor) of control group BCG bacterium cell wall label.Its
In, 1 is light field, and 2 be the channel FITC, and 3 be the channel TXRED, and 4 be 1,2,3 stacking chart.
Specific embodiment
The N- acetyl Portugal that the present invention is modified nitrine by the Biometabolic pathway of the peptide glycan in gram-positive bacterium
Osamine -1- phosphoric acid is introduced into the peptide glycan of gram positive bacterial cell wall, is drawn in a mild condition using bio-orthogonal reaction
Enter probe molecule (detectable marker), to realize the label to the gram positive bacterial cell wall of survival.Compared to making
With other labeling methods of genetic engineering, this method operates more simple.Due to the bone of whole gram positive bacterial cell walls
Frame is peptide glycan, and N-acetyl-glucosamine -1- phosphoric acid (GlcNAc-1P) is the synthesis precursor of peptide glycan, and this method is blue to leather
Family name's positive bacteria has universality.In addition, growth of the non-natural N-acetyl-glucosamine -1- phosphoric acid used in the present invention to bacterium
It is had little effect with activity, helps to realize quick, the convenient and fast label to gram-positive bacterium viable bacteria.
Term " gram-positive bacterium " used in the present invention has meaning well known in the art, that is, due to its cell
Wall main component can dye the bacterium of navy blue or purple for peptide glycan by Gram's staining.Present invention side can be used as
The gram-positive bacterium of method test object is the whole bacteriums for meeting gram-positive bacterium class definition, including but unlimited
In：Mycobacterium (Mycobacterium), staphylococcus (Staphylococcus), enterococcus spp
(Enterococcus), fusobacterium (Clostridium), Listeria (Listeria), streptococcus
(Streptococcus), bacillus (Bacillus), Corynebacterium (Corynebacterium) etc..Preferably, described
Gram-positive bacterium is selected from：Mycobacterium tuberculosis (Mycobacterium tuberculosis), staphylococcus aureus
(Staphylococcus aureus), streptococcus pneumonia (Streptococcus pneumoniae), bacillus anthracis
(Bacillus anthracis), corynebacterium diphtheriae (Corynebacterium diphtheriae), clostridium tetani
(Clostridium tetani)。
Peptide glycan is the main ingredient for constituting gram positive bacterial cell wall, it is by N-acetyl-glucosamine (G) and N-
Small peptide made of acetylmuramic acid (M) and four amino acid condensations is formed.This research is modified using non-natural nitrine
GlcNAc-1-P derivative can enter the peptide glycan of cell wall via the Cell wall synthesis approach of bacterium living, realize to leather
The label of gram-positive bacteria cell wall.
The free hydroxyl group of the full acetylated N-acetyl-glucosamine -1- phosphoric acid of nitrine modification in the present invention is all by acetyl
Change, so that the efficiency that the non-natural sugar was transported film is higher.In fact, the azido can be connected to by any way
Any one carbon atom in the glucose hexatomic ring of full acetylated N-acetyl-glucosamine -1- phosphoric acid.By making non-natural phosphate Portugal
The azido of osamine and alkynyl or triaryl phosphine progress bio-orthogonal reaction with detectable marker, by detectable marker
(that is, probe) is coupled to the peptide glycan of cell wall.
Preferably, the full acetylated N-acetyl-glucosamine -1- phosphoric acid of the nitrine modification is (2R, 3R, 4R, 5R, 6R) -5-
Acetylaminohydroxyphenylarsonic acid 2- (azido methyl) -6- (phosphono oxygroup) -3,4- diacetoxy-tetrahydro -2H- pyrans ((2R, 3R, 4R,
5R,6R)-5-acetamido-2-(azidomethyl)-6-(phosphonooxy)tetrahydro-2H-pyran-3,4-
Diyl diacetate) (referred to herein simply as 6N3- AcOGlcNAc-1P), structure is as shown in Equation 1.6N3-AcOGlcNAc-
1P synthetic method is known in the art dawn, and commercially available, for example, can through the invention in step described in embodiment 1 close
At.
Wherein, Ac is acetyl group, N3For azido group.
In the method for the invention, nitrine modification full acetylated N-acetyl-glucosamine -1- phosphoric acid can be connected with alkynes
The detectable marker of base or triaryl phosphine reacts, so that detectable marker to be coupled to the non-natural osamine of peptide glycan
(that is, full acetylated N-acetyl-glucosamine -1- phosphoric acid of nitrine modification) residue.
Detectable marker as described herein includes but is not limited to radioactive isotope, chromophore, antibody, chemiluminescence
Close object, spectral colorimetric mark object, fluorescent chemicals, metallo-chelate and enzyme.
Detectable marker used in method described herein can be primary marker (wherein, marker include can
The part for the signal that the part directly measured or generation can be measured directly) or secondary marker (wherein, detectable marker
Another reagent is bound to generate measurable signal).Detectable marker can be connected to by means covalently or non-covalently
Alkynyl/triaryl phosphine part.For example, detectable marker may be connected directly to alkynyl/triaryl phosphine part；Alternatively, detectable
Marker by ligand-receptor combine to, biotin-avidin (such as Streptavidin or ovum Avidin) gametophyte pair or its
Its such specific recognition molecules realize the connection with alkynyl or triaryl phosphine part.
In some embodiments, detectable marker can be fluorescent chemicals, such as luminescent dye molecule or fluorogen,
Including but not limited to：Fluorescein, such as 6- Fluoresceincarboxylic acid, 6- carboxyl -4 ', 5 '-two chloro- 2 ', 7 '-dimethoxyfluoresceins,
Fluorescein isothiocynate (FITC)；Rhodamine and its derivative, such as N, N, N ', -6 carboxyrhodamine (TAMRA of N '-tetramethyl
Or T), 6- carboxy-X-rhodamine (ROX or R), 5- carboxyrhodamine -6G (R6G5 or G5), 6- carboxyrhodamine -6G (R6G6 or
G6), rhodamine 110, four rhodamine isothiocyanates (tetrarhodimine isothiocynate, TRITC)；Cyan uniformly dyeing
Material, such as Cy3, Cy5 and Cy7 dyestuff；Cumarin, such as umbelliferone etc..Preferably, the fluorescent chemicals are N, N, N ', N '-
- 6 carboxyrhodamine of tetramethyl.
In some embodiments, detectable marker can be enzyme, and the enzyme as marker can produce for example chemical
Luminous signal, colourful signal or fluorescence signal, such as Fluc, Renilla luciferase.Alternatively, for detectably
The enzyme of labelled antibody reagent includes but is not limited to：Malic dehydrogenase, staphylococcal nuclease, δ-V- steroids heterologous enzyme,
Alcohol Dehydrogenase from Yeast, alpha-phosphate glycerol dehydrogenase, phosphotriose isomerase, horseradish peroxidase, alkaline phosphatase, asparagus fern acyl
Amine enzyme, glucose oxidase, beta galactosidase, ribalgilase, urase, catalase, glucose-VI-phosphate dehydrogenation
Enzyme, glucoamylase and acetylcholinesterase.
In some embodiments, detectable marker is chemiluminescence compound, including but not limited to：Lucigenin, Shandong
Minot, (adamantane) -1,2- dichloroethane, different luminol, imidazoles, acridinium ester, acridinium carboxamide, tris (bipyridine) ruthenium and oxalate.
In some embodiments, can measure marker can be spectral colorimetric mark object, including but not limited to：Colloidal gold
Or coloured glass or plastics (for example, polystyrene, polypropylene and latex) pearl.
In some embodiments, detectable marker can be radioactive isotope, including but not limited to：3H、125I、35S、14C、32P and33P。
In some embodiments, detectable marker includes any marker that can be determined by：Spectrum means；
Photochemistry means；Biochemistry means；Immunochemistry means；Electromagnetic means；Radiochemistry means；Or chemical means, such as it is glimmering
Light means, chemiluminescence means or chemiluminescence means or any other means appropriate.
In a preferred embodiment, detectable marker via biotin-avidin gametophyte to be connected to alkynyl or
Triaryl phosphine, the alkynyl or triaryl phosphine and azido form bio-orthogonal reaction group pair.
Preferably, the detectable marker for being connected with alkynyl is selected from the group as composed by following substance：
Wherein, describedIt is the detectable marker for being connected directly or indirectly to alkynyl moiety.
Preferably, the detectable marker for being connected with triaryl phosphine is selected from the group as composed by following substance：
Wherein, describedIt is the detectable marker for being connected directly or indirectly to triaryl phosphine part.
The synthetic method of above compound is known in the art or is obtained commercially.
In some preferred embodiments, alkynyl/triaryl phosphine detectable marker that is connected with is by containing
Dibenzazepine cyclo-octene-PEG4- biotin (the Dibenzocyclooctyne-PEG4-biotin of high-tension alkynes
Conjugate (DBCO-PEG4-biotin)) with Streptavidin-detectable marker (that is, be connected with Streptavidin can
Detect marker) conjugation acquisition.Shown in the structure such as formula (II-7) of the dibenzazepine cyclo-octene-PEG4- biotin：
In other preferred embodiments, the detectable marker for being connected with alkynyl or triaryl phosphine be containing
(Dibenzocycl-ooctyne (DBCO)-PEG4- of dibenzazepine cyclo-octene-PEG4- fluorescein 545 of power alkynes
Fluor545 or DBCO-PEG4-TAMRA), shown in structure such as formula (II-8).
Above-mentioned whole compound and a variety of detectable markers (radioactive isotope, hair for being modified with Streptavidin
Color group, antibody, chemiluminescence compound, spectral colorimetric mark object, fluorescent chemicals, metallo-chelate and enzyme) it is commercially available
It arrives.
Method of the invention can realize the label to gram-positive bacterium (such as mycobacterium tuberculosis), including walk as follows
Suddenly：
(i) the full acetylated N-acetyl-glucosamine -1- phosphoric acid of nitrine modification is added in gram-positive bacterium culture medium,
Gram-positive bacterium is inoculated in the culture medium and the gram-positive bacterium is cultivated, obtains Bacteria Culture
Object；
(ii) bacterial cultures that collection step (i) obtains obtains the precipitating of the bacterial cultures by processing
Object；
(iii) bacterium in the sediment obtained to step (ii) is fixed；
(iv) to the detectable label for being connected with alkynyl or triaryl phosphine through adding in fixed bacterium of step (iii)
Object, so that the marker is coupled to the peptide glycan in the bacterium；
(v) detectable marker is detected.
Inoculum density, condition of culture, incubation time of the gram-positive bacterium of step (i) etc. are this field for thin
The normal condition or parameter of bacterium culture.
Preferably, the full acetylated N-acetyl-glucosamine -1- phosphoric acid of the nitrine modification of step (i) is the formula (I)
Compound.Preferably, by the compound of the formula (I) with 120-200 μ g/mL, the final concentration of preferably 160 μ g/mL is added to
Culture medium.
It is described to be collected as routine operation in step (ii), for example, sterile water washing bacterium can be used for solid culture
The bacterial cultures that bacteria suspension is obtained as collection is come to, or liquid culture can be collected directly into centrifuge tube
Deng.Preferably, such as liquid culture pass through centrifugation after collection and obtain sediment.Further preferably, slow using PBSTB
The sediment for the bacterial cultures that fliud flushing is washed washes away unemployed non-natural compound (that is, the full second of nitrine modification
Acylated N-acetyl-glucosamine -1- phosphoric acid).In addition, the centrifugation is the routine operation of this field, those skilled in the art can root
According to the ordinary skill knowledge that it is grasped for the centrifugal rotational speed etc. that different gram-positive bacterium selections is suitable.
Preferably, in step (iii), bacterium is fixed using 4w/v% paraformaldehyde.Further preferably, in fixation
After be centrifuged, obtain bacterial precipitation and washed with PBSTB buffer, to wash away extra paraformaldehyde fixer.
It is preferred that before carrying out step (iv), by biotin-alkynyl moiety or biotin-triaryl phosphine part and strepto-
Avidin-detectable marker is coupled, and the detectable marker of alkynyl or triaryl phosphine is connected with described in formation.Alternatively, into
When row step (iv), to step (iii) through adding biotin-alkynyl moiety or biotin-triaryl phosphine in fixed cell
Streptavidin-detectable marker is then added in part, so that the marker is coupled to the intracellular sugared egg
It is white.
Preferably, the alkynyl or the detectable marker of triaryl phosphine of being connected with of step (iv) is the formula (II-8)
Compound.
It is not intended to be limited by theory ground, in above-mentioned reaction, being impregnated in the azido concentration of peptide glycan, determine can be by
In conjunction with the amount of formula (II-8) compound therefore azido is marked as long as guaranteeing that dosage can satisfy, without
Especially limitation.Preferably, the dosage of formula (II-8) compound is greater than 5mM.It is further preferred that described through fixed thin
The compound of the formula (II-8) of 10mM is added in bacterium.It is preferred that described through adding the formula (II-8) in fixed bacterium
Compound after be incubated for 1.5h.
The embodiment of various aspects described herein can be by the paragraph explanation numbered as follows：
1. a kind of method that the gram-positive bacterium to survival is marked, described method includes following steps：
(i) the full acetylated N-acetyl-glucosamine -1- phosphoric acid of nitrine modification is added in gram-positive bacterium culture medium,
Gram-positive bacterium is inoculated in the culture medium and the gram-positive bacterium is cultivated, obtains Bacteria Culture
Object；
(ii) bacterial cultures that collection step (i) obtains obtains the precipitating of the bacterial cultures by processing
Object；
(iii) bacterium in the sediment obtained to step (ii) is fixed；
(iv) to the detectable label for being connected with alkynyl or triaryl phosphine through adding in fixed bacterium of step (iii)
Object, so that the marker is coupled to the peptide glycan in the bacterium；
(v) detectable marker is detected.
2. the method as described in paragraph 1, wherein the full acetylated N-acetyl-glucosamine -1- phosphoric acid of nitrine modification is
(2R, 3R, 4R, 5R, 6R) -5- acetylaminohydroxyphenylarsonic acid 2- (azido methyl) -6- (phosphono oxygroup) -3,4- diacetoxy-four
Hydrogen -2H- pyrans.
3. the method as described in paragraph 1 or 2, wherein the detectable marker for being connected with alkynyl is selected from by as follows
Group composed by substance：
Wherein, the detectable marker for being connected with triaryl phosphine is selected from the group as composed by following substance：
Wherein, describedIt is the detectable marker for being connected directly or indirectly to alkynyl moiety or triaryl phosphine part.
4. the method as described in any one of paragraph 1-3, wherein the detectable marker is selected from：The same position of radioactivity
Element, chromophore, antibody, chemiluminescence compound, spectral colorimetric mark object, fluorescent chemicals, metallo-chelate and enzyme.
5. the method as described in paragraph 4, wherein the radioactive isotope is selected from：
3H、125I、35S、14C、32P and33P。
6. the method as described in paragraph 4, wherein the fluorescent chemicals are selected from：
Fluorescein, such as 6- Fluoresceincarboxylic acid, 6- carboxyl -4 ', 5 '-two chloro- 2 ', 7 '-dimethoxyfluoresceins, different sulphur cyanogen
Sour fluorescein)；Rhodamine and its derivative, such as N, N, N ', -6 carboxyrhodamine of N '-tetramethyl, 6- carboxy-X-rhodamine,
5- carboxyrhodamine -6G, 6- carboxyrhodamine -6G, rhodamine 110, four rhodamine isothiocyanates；Cyanine dye, such as
Cy3, Cy5 and Cy7 dyestuff；Cumarin, such as umbelliferone etc.；
Preferably, the fluorescent chemicals are N, N, N ', -6 carboxyrhodamine of N '-tetramethyl.
7. the method as described in paragraph 4, wherein the enzyme is selected from Fluc, Renilla luciferase.
8. the method as described in paragraph 4, wherein the enzyme is selected from：
Malic dehydrogenase, staphylococcal nuclease, δ-V- steroids heterologous enzyme, Alcohol Dehydrogenase from Yeast, alpha-phosphate are sweet
Oily dehydrogenase, phosphotriose isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-
Galactosidase, ribalgilase, urase, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetyl
Cholinesterase.
9. the method as described in paragraph 4, wherein the chemiluminescence compound is selected from：
Lucigenin, luminol, (adamantane) -1,2- dichloroethane, different luminol, imidazoles, acridinium ester, acridinium carboxamide, three
Bipyridyl ruthenium and oxalate.
10. the method as described in paragraph 4, wherein the spectral colorimetric mark object is selected from：
Colloidal gold or coloured glass or plastic bead.
11. the method as described in any one of paragraph 1-10, wherein the detectable marker is connected directly to the alkynes
Base portion point or triaryl phosphine part, to be connected with the detectable marker of alkynyl or triaryl phosphine described in being formed.
12. the method as described in any one of paragraph 1-10, wherein the detectable marker is via ligand-receptor knot
Conjunction pair or biotin-avidin gametophyte are to the alkynyl moiety or triaryl phosphine part is connected to, to form the connection
There is the detectable marker of alkynyl or triaryl phosphine.
13. the method as described in paragraph 12, wherein the Avidin of the biotin-avidin gametophyte centering is strepto-
Avidin or ovum Avidin.
14. the method as described in paragraph 13, wherein before carrying out step (iv), by biotin-alkynyl moiety or biology
Element-triaryl phosphine part and Streptavidin-detectable marker are coupled, and be connected with alkynyl or triaryl phosphine described in formation can
Detect marker.
15. the method as described in paragraph 13, wherein when carrying out step (iv), to step (iii) through fixed cell
Streptavidin-detectable marker is then added in middle addition biotin-alkynyl moiety or biotin-triaryl phosphine part, from
And the marker is coupled to the intracellular glycoprotein.
16. the method as described in paragraph 14 or 15, wherein the biotin-alkynyl moiety is dibenzazepine cyclo-octene-
PEG4- biotin.
17. the method as described in paragraph 4, wherein the alkynyl or the detectable marker of triaryl phosphine of being connected with is two
Benzo-aza cyclo-octene-PEG4- fluorescein 545.
18. the method as described in any one of paragraph 1-17, wherein in step (ii), washed using PBSTB buffer
The sediment of the bacterial cultures.
19. the method as described in any one of paragraph 1-18, wherein in step (iii), using paraformaldehyde to described
Bacterium is fixed；It is preferred that being centrifuged to described through fixed bacterium after the fixation, obtains cell precipitation and use PBSTB
Buffer is washed.
20. the method as described in any one of paragraph 2-18, wherein (2R, 3R, 4R, 5R, 6R) -5- acetyl ammonia by described in
Base -2- (azido methyl) -6- (phosphono oxygroup) -3,4- diacetoxy-tetrahydro -2H- pyrans is with 120-200 μ g/mL, excellent
The final concentration of 160 μ g/mL is selected to be added to the culture medium.
21. the method as described in any one of paragraph 1-20, wherein the method is used for the thin of gram-positive bacterium
Cell wall is marked.
22. the method as described in any one of paragraph 1-21, wherein the gram-positive bacterium is selected from：
Mycobacterium, staphylococcus, enterococcus spp, fusobacterium, Listeria, streptococcus, bacillus
Belong to, Corynebacterium.
23. the method as described in any one of paragraph 1-21, wherein the gram-positive bacterium is selected from：
Mycobacterium tuberculosis, staphylococcus aureus, streptococcus pneumonia, bacillus anthracis, corynebacterium diphtheriae, clostridium tetani.
24. the full acetylated N-acetyl-glucosamine -1- phosphoric acid of nitrine modification is used for the gram-positive bacterium of survival
The purposes that cell wall is marked, wherein pass through the nitrine for the full acetylated N-acetyl-glucosamine -1- phosphoric acid that the nitrine is modified
The bio-orthogonal reaction of base and the alkynyl or triaryl phosphine that are connected to detectable marker realizes the Gram-positive to survival
The label of the cell wall of bacterium.
25. the full acetylated N-acetyl-glucosamine -1- phosphoric acid of nitrine modification is used for specific in the sample comprising bacterium
The purposes of the gram-positive bacterium of ground label survival, wherein the full acetylated N-acetyl-glucosamine-modified by the nitrine
The azido of 1- phosphoric acid and the bio-orthogonal reaction of the alkynyl or triaryl phosphine that are connected to detectable marker are realized to survival
Gram-positive bacterium cell wall label.
26. the purposes as described in paragraph 24 or 25, wherein the full acetylated N-acetyl-glucosamine -1- of the nitrine modification
Phosphoric acid is (2R, 3R, 4R, 5R, 6R) -5- acetylaminohydroxyphenylarsonic acid 2- (azido methyl) -6- (phosphono oxygroup) -3,4- diethyl acyl-oxygen
Base-tetrahydro -2H- pyrans.
27. the purposes as described in any one of paragraph 24-26, wherein the detectable marker is selected from：Radioactivity is same
Position element, chromophore, antibody, chemiluminescence compound, spectral colorimetric mark object, fluorescent chemicals, metallo-chelate and enzyme.
28. the purposes as described in paragraph 27, wherein the radioactive isotope is selected from：
3H、125I、35S、14C、32P and33P。
29. the purposes as described in paragraph 27, wherein the fluorescent chemicals are selected from：
Fluorescein, such as 6- Fluoresceincarboxylic acid, 6- carboxyl -4 ', 5 '-two chloro- 2 ', 7 '-dimethoxyfluoresceins, different sulphur cyanogen
Sour fluorescein)；Rhodamine and its derivative, such as N, N, N ', -6 carboxyrhodamine of N '-tetramethyl, 6- carboxy-X-rhodamine,
5- carboxyrhodamine -6G, 6- carboxyrhodamine -6G, rhodamine 110, four rhodamine isothiocyanates；Cyanine dye, such as
Cy3, Cy5 and Cy7 dyestuff；Cumarin, such as umbelliferone etc.；
Preferably, the fluorescent chemicals are N, N, N ', -6 carboxyrhodamine of N '-tetramethyl.
30. the purposes as described in paragraph 27, wherein the enzyme is selected from Fluc, Renilla luciferase.
31. the purposes as described in paragraph 27, wherein the enzyme is selected from：
Malic dehydrogenase, staphylococcal nuclease, δ-V- steroids heterologous enzyme, Alcohol Dehydrogenase from Yeast, alpha-phosphate are sweet
Oily dehydrogenase, phosphotriose isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-
Galactosidase, ribalgilase, urase, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetyl
Cholinesterase.
32. the purposes as described in paragraph 27, wherein the chemiluminescence compound is selected from：
Lucigenin, luminol, (adamantane) -1,2- dichloroethane, different luminol, imidazoles, acridinium ester, acridinium carboxamide, three
Bipyridyl ruthenium and oxalate.
33. the method as described in paragraph 27, wherein the spectral colorimetric mark object is selected from：
Colloidal gold or coloured glass or plastic bead.
34. a kind of for detecting the kit of the gram-positive bacterium of survival, wherein the kit is repaired comprising nitrine
Full acetylated N-acetyl-glucosamine -1- the phosphoric acid of decorations and the detectable marker for being connected with alkynyl or triaryl phosphine.
35. the kit as described in paragraph 34, wherein the full acetylated N-acetyl-glucosamine -1- phosphorus of the nitrine modification
Acid is (2R, 3R, 4R, 5R, 6R) -5- acetylaminohydroxyphenylarsonic acid 2- (azido methyl) -6- (phosphono oxygroup) -3,4- diacetoxy -
Tetrahydro -2H- pyrans.
36. the kit as described in paragraph 34 or 35, wherein the detectable marker for being connected with alkynyl be selected from by
Group composed by following substance：
Wherein, the detectable marker for being connected with triaryl phosphine is selected from the group as composed by following substance：
Wherein, describedIt is the detectable marker for being connected directly or indirectly to alkynyl moiety or triaryl phosphine part.
37. the kit as described in any one of paragraph 34-36, wherein the detectable marker is selected from：Radioactivity
Isotope, chromophore, antibody, chemiluminescence compound, spectral colorimetric mark object, fluorescent chemicals, metallo-chelate and enzyme.
38. the kit as described in paragraph 37, wherein the radioactive isotope is selected from：
3H、125I、35S、14C、32P and33P。
39. the kit as described in paragraph 37, wherein the fluorescent chemicals are selected from：
Fluorescein, such as 6- Fluoresceincarboxylic acid, 6- carboxyl -4 ', 5 '-two chloro- 2 ', 7 '-dimethoxyfluoresceins, different sulphur cyanogen
Sour fluorescein)；Rhodamine and its derivative, such as N, N, N ', -6 carboxyrhodamine of N '-tetramethyl, 6- carboxy-X-rhodamine,
5- carboxyrhodamine -6G, 6- carboxyrhodamine -6G, rhodamine 110, four rhodamine isothiocyanates；Cyanine dye, such as
Cy3, Cy5 and Cy7 dyestuff；Cumarin, such as umbelliferone etc.；
Preferably, the fluorescent chemicals are N, N, N ', -6 carboxyrhodamine of N '-tetramethyl.
40. the kit as described in paragraph 37, wherein the enzyme is selected from Fluc, Renilla luciferase.
41. the kit as described in paragraph 37, wherein the enzyme is selected from：
Malic dehydrogenase, staphylococcal nuclease, δ-V- steroids heterologous enzyme, Alcohol Dehydrogenase from Yeast, alpha-phosphate are sweet
Oily dehydrogenase, phosphotriose isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-
Galactosidase, ribalgilase, urase, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetyl
Cholinesterase.
42. the kit as described in paragraph 37, wherein the chemiluminescence compound is selected from：
Lucigenin, luminol, (adamantane) -1,2- dichloroethane, different luminol, imidazoles, acridinium ester, acridinium carboxamide, three
Bipyridyl ruthenium and oxalate.
43. the kit as described in paragraph 37, wherein the spectral colorimetric mark object is selected from：
Colloidal gold or coloured glass or plastic bead.
Embodiment
Tuberculosis is the chronic infectious disease as caused by bacillus tuberculosis typus humanus (M.tuberculosis), is world's model
The interior second largest communicable disease is enclosed, the drug target for treating tuberculosis at present is much Mycobacterium tuberculosis cell wall.In the present invention
Embodiment in, we using with the homologous mycobacterium tuberculosis var bovis of bacillus tuberculosis typus humanus as illustrative gram to be detected
Positive bacteria is marked its cell wall using method of the invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.Such as without spy
Do not mentionlet alone it is bright, whole reagents be purchased from Sigma-Aldrich.
Agents useful for same in embodiment：
7H9 culture medium：0.94g 7H9 culture medium powder, 0.4mL glycerol, 0.1mL are added in 180ml distilled water
Tween80,121 DEG C of sterilizing 10min.Use preceding increasing microbial inoculum OADC of the addition 20mL through filtration sterilization.
OADC：0.255g NaCl, 1.5g BSA-V, 0.6g glucose, 0.016g oleic acid are added in 30mL distilled water
Sodium, 0.0012g catalase (catalase), filtration sterilization.
PBS buffer solution (10mM)：Solvent is water, solute NaH2PO4、Na2HPO4, KCl and NaCl, the solute
NaH2PO4、Na2HPO4, the concentration of KCl and NaCl in the PBS buffer solution be respectively 0.24g/L, 1.42g/L, 0.2g/L and
8.0g/L；The pH value of the PBS buffer solution is 7.4.
PBSTB buffer (10mM)：Solvent is water, solute NaH2PO4、Na2HPO4, KCl, NaCl, BSA and polysorbas20,
The solute NaH2PO4、Na2HPO4, the concentration of KCl, NaCl, BSA and polysorbas20 in the PBS buffer solution be respectively
0.24g/L, 1.42g/L, 0.2g/L, 8.0g/L, 0.5w/v% and 0.1v/v%.The pH value of the PBSTB buffer is 7.4.
PB buffer solution：Solvent is water, solute NaH2PO4、Na2HPO4, the solute NaH2PO4、Na2HPO4Described
Concentration in PBS buffer solution is respectively 0.24g/L and 1.42g/L.The pH value of the PB buffer is 7.4.
TCA solution：10g trichloroacetic acid is dissolved in 100mL water, with magnetic stirrer until being completely dissolved.
Combination buffer (PH 7.5)：Solvent is water, solute Tris, NaCl.Described solute Tris, NaCl are in the knot
The concentration for closing buffer is respectively 2.4g/L, 2.9g/L；The pH value of the combination buffer is 7.5.
545 (the Dibenzocycl-ooctyne of dibenzazepine cyclo-octene-PEG4- fluorescein of the alkynes containing tension
(DBCO)-PEG4-Fluor545) it is purchased from Sigma-Aldrich (article No. 760773).
Mycobacterium tuberculosis var bovis (Latin entitled Mycobacterium bovis, entitled Bacillus generally in the art
Calmette–Guérin)：Bacterial strain is BCG bacterial strain/BCG Pasteur 1173P2, which has pUV3583c-GFP plasmid,
GFP can be expressed.BCG bacterial strain/BCG Pasteur1173P2 building and explanation refer to Fungal
biotransformation of tanshinone results in[4+2]cycloaddition with
sorbicillinol:evidence for enzyme catalysis and increased antibacterial
Activity, Wenni He etc., Applied Microbiology and Biotechnology, 2016.
Embodiment 1 (2R, 3R, 4R, 5R, 6R) -5- acetylaminohydroxyphenylarsonic acid 2- (azido methyl) -6- (phosphono oxygroup) -3,4-
Diacetoxy-tetrahydro -2H- pyrans (6N3- AcOGlcNAc-1P) chemical synthesis
6N3- AcOGlcNAc-1P's synthesizes using nitrogen acetylglucosamine as starting material progress.Synthesis path is as follows：
Wherein, Ac is acetyl group, N3For azido group, TS is p-toluenesulfonyl, and Me is methyl.
10.35g is added to methyl in 10.00g nitrogen acetylglucosamine (0.045mol) (being purchased from Sigma, article No. A3286)
Benzene sulfonyl chloride (0.054mol) is stirred to react overnight with 300ml pyridinium dissolution.45ml acetic anhydride (0.528mol) reaction is added
6h is spin-dried for pyridine with oil pump, then uses 1M hydrochloric acid and saturated sodium bicarbonate extracting and washing respectively.Merge organic phase, uses anhydrous slufuric acid
Sodium is dry.After being spin-dried for, compound (mobile phase is separated using column phase flash chromatography：Ethyl acetate/petroleum ether=2:1) syrup is obtained
Shape compound 6c (12.00g, yield 70%).To addition 4.00g sodium azide in 10.00g compound 6c (0.026mol)
(0.059mol) is dissolved in 150ml dimethylformamide, and 65 DEG C are heated to reflux 12h, uses saturated sodium-chloride and acetic acid second after being spin-dried for again
Ester extraction, is dried, filtered with anhydrous sodium sulfate, is spin-dried for, and separates compound (mobile phase using column phase flash chromatography：Ethyl acetate/
Petroleum ether=1:1) compound 6d (7.20g, yield 75%), is obtained.Sodium methoxide/methanol (1M) is added and adjusts pH to 9, at room temperature
After reacting 3h, H is used+Ion exchange resin neutralizes, and filters, is spin-dried for, obtains compound as white solid 6e (4.40g, yield
93%).In 10ml reaction system, to addition 112mg ATP (20mM) and 7.5mg N- in 54.1mg compound 6e (22mM)
1- kinases (NahK) (0.75mg/mL) of acetylaminohexose；Buffer is 100mM Tris-HCl (MgCl containing 10mM2),
pH9.0；Reaction temperature is 37 DEG C, reaction time 12h.It boils 5min and stops reaction, 12000g is centrifuged 5min, collects supernatant
Gel column is crossed after outstanding inspissation contracting, compound 6f (64mg, yield 90%) is obtained, with anisaldehyde chromogenic reagent.By 100mg chemical combination
Object 6f (0.31mmol) is dissolved to 10ml pyridine, and acetic anhydride (0.92mmol) reaction 6h is slowly added dropwise under ice bath, dissolves after being spin-dried for
It in methylene chloride, is successively washed using 1M hydrochloric acid and saturated sodium bicarbonate, merging organic phase is simultaneously dry with anhydrous sodium sulfate.It is spin-dried for
Afterwards, compound (mobile phase is separated using column phase flash chromatography：Ethyl acetate/methanol=1:1) compound as white solid 6 is obtained
(95mg, yield 75%), i.e. 6N3- AcOGlcNAc-1P, shown in structural formula such as formula (I).
6 Structural Identification of compound is as follows：1H NMR(500MHz,D2O):δ=5.38 (dd, 1H, J=7.1Hz, 3.2Hz, H-
1), 5.23 (t, 1H, J=9.5Hz), 5.07 (t, 1H, J=9.7Hz), 4.23 (dd, 2H, J=21.05,10.55Hz), 3.64-
3.56(m,1H),3.39-3.36(m,1H),2.01(s,3H),1.95(s,3H),1.90(s,3H).HRESIMS Caled for
C12H19N4O10P(M-H)-:409.0826；found m/z 409.0749.
The extraction of 2 mycobacterium tuberculosis peptide glycan of embodiment
In order to prove that formula (I) compound can be inserted into the peptide glycan of cell wall through bacterial metabolism, we are by formula (I)
Compound is incubated for jointly with mycobacterium tuberculosis var bovis, extracts its peptide glycan, or discharge monosaccharide with bacteriolyze enzymatic treatment, then use
HPLC-MS analyzes the ingredient of peptide glycan or monosaccharide.Wherein, lysozyme can act on the N- acetyl of peptide glycan molecule in specific manner
β-Isosorbide-5-Nitrae glycosidic linkage between muramic acid and N-acetyl-glucosamine is allowed to fracture and obtains monosaccharide.
(1) BCG bacterium is inoculated in respectively in two bottles of 2L glycerol alanine culture mediums, 37 DEG C are slightly shaken 10 days, keep BCG bacterium raw
It grows to mid-log phase.The formula (I) of the acquisition of embodiment 1 of final concentration of 160 μ g/mL is added in wherein one bottle of cell culture
Compound (experimental group), another bottle are added without any compound as negative control (control group), continue culture 7 days.
(2) bacterium of culture is centrifuged with 12000g revolving speed, and is rinsed with PBS buffer solution, 100 DEG C of high temperature are boiled inactivation.
With 0.22 μm of membrane filtration, bacterial cell is separated with culture medium, obtain 9g bacterial cell (weight in wet base), with 4.5ml TSE (10mM
Tris-HCl, 150mM NaCl and 1mM EDTA) cell is resuspended, 4 DEG C of high pressures of instrument (JNBIO-Mini) are crushed with cryogenic high pressure
(1500bar) is broken, will be centrifuged 20min in 4 DEG C, 2500g through broken cell suspending liquid, removes unbroken cell and broken
Piece collects supernatant, in 4 DEG C, 27000g, is centrifuged 1h, obtains cell wall.
(3) after the dodecyl sodium sulfate (SDS) of 8w/v% is added into cell wall, boiling water bath boils 10min, is cooled to
After room temperature, 12000r/min is centrifuged 15min and collects precipitating.It is washed repeatedly with PBS buffer solution until being precipitated to non-foam.It will obtain
Precipitating be added to 0.lmol/L, pH value 7.8 PB buffer solution (trypsase containing 3mg/ml) in handle 3h, then will
Mixed liquor collects supernatant in 3000r/min centrifugation 5min.Gained supernatant 12000r/min centrifugation 15min is collected into precipitating.
(4) the resulting precipitating TCA solution of 10g/dL last in step (3) is resuspended, it is cold after 90 DEG C of water-bath 20min
But to room temperature.12000r/min is centrifuged 15min, collects precipitating, is washed with distilled water three times.
(5) precipitating that step (4) obtain is resuspended with ether, 12000r/min is centrifuged 15min again after standing 30min, receives
Collection precipitating, is dehydrated, 70 DEG C of dryings with dehydrated alcohol, obtains peptide glycan.
(6) bacterium in the PBS of 3.5ml by step (1) culture dilutes, ultrasonotomography.Mutanolysin (is bought from Sa
Grace chemical technology Co., Ltd, article No.：A07M9901) (purchase is dissolved to 500 from magnificent Boulder hundred million, article No. CL6951) with lysozyme
In the Tris-HCl (0.5mM, pH6.5) of μ l, its final concentration is made to be respectively 150 μ g/ml and 100 μ g/ml, then and ultrasonotomography
The fragment of acquisition is slowly mixed together, and incubates 4h at 37 DEG C.Sample is heated 5 minutes at 90 DEG C, lysozyme and bacteriolyze enzyme-deactivating will be become.
(7) it is carried out using appearance situation of the HPLC-MS to the monosaccharide of step (5) peptide glycan obtained and step (6) acquisition
Detection.
HPLC condition is as follows：Using Nova-Pak C18 (3.9 × 150mm, 4 μm) chromatographic column, with 20mM triethylamine-acetic acid
It (pH4.0) is mobile phase, flow velocity 0.5mL/min, Detection wavelength 260nm, column temperature is room temperature.
Fig. 1 is the HPLC map of experimental group peptide glycan.Fig. 2 is 6- azido methyl-N- Acetylglucos in experimental group monosaccharide
The LC-MS map of amine.Fig. 3 is the LC-MS map of N-acetyl-glucosamine in experimental group.Experimental group is extracted it can be seen from Fig. 1-3
Peptide glycan can find two obvious peaks in monosaccharide molecule amount range：Nitrogen acetylglucosamine (Glc-NAc, M=221.08) and
6- azido methyl-N-acetyl-glucosamine (6N3- GlcNAc, M=246.10).
Fig. 4 is the HPLC map of BCG bacterium peptide glycan in control group.Fig. 5 is the LC-MS figure of nitrogen acetylglucosamine in control group
Spectrum.By Figure 4 and 5 it is found that when formula (I) compound that embodiment 1 obtains is not added in the medium, the BCG bacterium institute cultivated
The peptide glycan of extraction only finds an obvious peak in monosaccharide molecule amount range：N-acetyl-glucosamine (Glc-NAc, M=
221.08), without 6N3The peak of-GlcNAc.
The compound of the provable formula of result above (I) can be inserted into peptide glycan.
The label of 3 Mycobacterium tuberculosis cell wall of embodiment
(i) prepare two bottles of 2L glycerol alanine culture mediums, what one bottle of embodiment 1 that final concentration of 160 μ g/mL is added obtained
The compound (experimental group) of formula (I), another bottle are added without any compound as negative control (control group), it is raw will to be in logarithm
Long-term mycobacterium tuberculosis (BCG bacterium) is inoculated in above-mentioned culture medium respectively, and 37 DEG C are cultivated 14 days, obtains cell culture fluid.
(ii) cell suspension that collection step (i) obtains, 12000g centrifugation, obtains the precipitating of the cell；It is used after centrifugation
PBSTB buffer rinses bacterium precipitating 3 times.
(iii) cell in the precipitating obtained with 4w/v% formaldehyde to step (ii) fixes 20min, then uses PBSTB
Buffer rinses three times.
(iv) it is incubated at room temperature to step (iii) through adding 10mM DBCO-PEG4-Fluor545 in fixed cell
1.5h, so that red fluorescence marker to be coupled to the peptide glycan in bacterium.
(v) detectable marker is detected using Laser Scanning Confocal Microscope.
As a result as shown in figs 6-8.
Fig. 6 is the fluorescence photo of the label of experimental group BCG bacterium cell wall under 100 times of mirrors.As shown in Figure 6, wherein (1) be
The form of the BCG bacterium observed under light field；(2) for the channel FITC as a result, display BCG bacterium expression GFP (absorption peak 395nm,
Emission peak 509nm) distribution；It (3) is the channel TXRED as a result, display Fluor545 (absorption peak 545nm, emission peak 567nm)
Distribution；It (4) is the stacking chart of (1)-(3).It can be seen that BCG bacterium surface there are red fluorescence (Fig. 6 (3)), illustrate formula (I)
Compound is by pass flag on the cell wall of mycobacterium tuberculosis.
Fig. 7 is the enlarged drawing (100 times of amplification factor) of a bacterium in Fig. 6.Wherein, (1) is the BCG observed under light field
The form of bacterium；It (2) is the channel FITC as a result, point of the GFP (absorption peak 395nm, emission peak 509nm) of display BCG bacterium expression
Cloth；It (3) is the channel TXRED as a result, the distribution of display Fluor545 (absorption peak 545nm, emission peak 567nm)；(4) it is (1)-
(3) stacking chart.It can be seen that GFP and Fluor545 common location, illustrate that the compound of formula (I) is divided by pass flag in tuberculosis
On the cell wall of branch bacillus.
Fig. 8 is the fluorescence photo that control group BCG bacterium cell wall marks under 100 times of mirrors.As shown in Figure 8, wherein (1) be bright
The form of the BCG bacterium observed off field；It (2) is the channel FITC as a result, GFP (absorption peak 395nm, the hair of display BCG bacterium expression
Penetrate peak 509nm) distribution；It (3) is the channel TXRED as a result, showing Fluor545 (absorption peak 545nm, emission peak 567nm)
Distribution；It (4) is the stacking chart of (1)-(3).It can be seen that illustrating that cell wall is not labeled without any signal in figure (3).
Above-described embodiment demonstrates the full acetylated N- of analog-nitrine modification of peptide glycan synthesis precursor GlcNAc-1P
Acetylglucosamine -1- phosphoric acid can be utilized by the GlmU enzyme of mycobacterium tuberculosis itself, and Mycobacterium tuberculosis cell has been arrived in metabolism
On wall, so as to be used to realize the label to the Mycobacterium tuberculosis cell wall of survival.
Claims (4)
1. a kind of method that the gram-positive bacterium to survival is marked, described method includes following steps：
(i) the full acetylated N-acetyl-glucosamine -1- phosphoric acid that nitrine modification is added in gram-positive bacterium culture medium, will remove from office
Gram-positive bacteria is inoculated in the culture medium and cultivates the gram-positive bacterium, obtains bacterial cultures；
(ii) bacterial cultures that collection step (i) obtains obtains the sediment of the bacterial cultures by processing；
(iii) bacterium in the sediment obtained to step (ii) is fixed；
(iv) detectable marker of alkynyl or triaryl phosphine is connected with through adding in fixed bacterium to step (iii), from
And the marker is coupled to the peptide glycan in the bacterium；
(v) detectable marker is detected.
2. the full acetylated N-acetyl-glucosamine -1- phosphoric acid of nitrine modification is used for the cell wall to the gram-positive bacterium of survival
The purposes being marked, wherein the azido for the full acetylated N-acetyl-glucosamine -1- phosphoric acid modified by the nitrine and company
The bio-orthogonal reaction of the alkynyl or triaryl phosphine that are connected to detectable marker is realized to the gram-positive bacterium of survival
The label of cell wall.
3. the full acetylated N-acetyl-glucosamine -1- phosphoric acid of nitrine modification in the sample comprising bacterium for specifically marking
The purposes of the gram-positive bacterium of survival, wherein the full acetylated N-acetyl-glucosamine -1- phosphoric acid modified by the nitrine
Azido and the alkynyl or triaryl phosphine that are connected to detectable marker bio-orthogonal reaction it is blue to the leather of survival to realize
The label of the cell wall of family name's positive bacteria.
4. a kind of for detecting the kit of the gram-positive bacterium of survival, wherein the kit includes nitrine modification
Full acetylated N-acetyl-glucosamine -1- phosphoric acid and the detectable marker for being connected with alkynyl or triaryl phosphine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710321663.7A CN108865918B (en) | 2017-05-09 | 2017-05-09 | Method for marking viable gram-positive bacteria and cell walls thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710321663.7A CN108865918B (en) | 2017-05-09 | 2017-05-09 | Method for marking viable gram-positive bacteria and cell walls thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108865918A true CN108865918A (en) | 2018-11-23 |
| CN108865918B CN108865918B (en) | 2022-01-07 |
Family
ID=64287473
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710321663.7A Expired - Fee Related CN108865918B (en) | 2017-05-09 | 2017-05-09 | Method for marking viable gram-positive bacteria and cell walls thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108865918B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111733102A (en) * | 2020-06-30 | 2020-10-02 | 东南大学 | Gram-positive bacterium surface modification method based on tyrosinase catalysis and application thereof |
| WO2021015622A1 (en) * | 2019-07-25 | 2021-01-28 | Synaffix B.V. | Synthesis of 6-azido-6-deoxy-2-n-acetyl-hexosamine-nucleoside diphosphate |
| CN114790435A (en) * | 2021-01-25 | 2022-07-26 | 山东大学 | Bacillus subtilis engineering strain for synthesizing click-reaction glycosaminoglycan skeleton and construction method and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1331276A1 (en) * | 2002-01-28 | 2003-07-30 | Universita' degli studi di Bologna | Method and kit for detecting Gram-positive bacteria |
| JP2006075029A (en) * | 2004-09-07 | 2006-03-23 | Asahi Breweries Ltd | Method for detecting gram-positive bacteria |
| CN102313801A (en) * | 2011-06-03 | 2012-01-11 | 南开大学 | Metabolic labeling method of antibody and application of antibody in fluorescence detection |
| CN104039977A (en) * | 2012-01-18 | 2014-09-10 | 国家科学研究中心 | A method for specifically labeling living bacteria |
| CN104459125A (en) * | 2014-12-10 | 2015-03-25 | 中国科学院苏州生物医学工程技术研究所 | Method for rapidly detecting gram negative and positive bacteria |
| CN104749369A (en) * | 2013-12-31 | 2015-07-01 | 中国科学院深圳先进技术研究院 | Fluorescent labeling method for living organisms having cell membrane structures |
| CN105683389A (en) * | 2013-10-30 | 2016-06-15 | 国家科学研究中心 | Method for labeling specifically living bacteria comprising the use of modified monosaccharide compounds |
-
2017
- 2017-05-09 CN CN201710321663.7A patent/CN108865918B/en not_active Expired - Fee Related
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1331276A1 (en) * | 2002-01-28 | 2003-07-30 | Universita' degli studi di Bologna | Method and kit for detecting Gram-positive bacteria |
| JP2006075029A (en) * | 2004-09-07 | 2006-03-23 | Asahi Breweries Ltd | Method for detecting gram-positive bacteria |
| CN102313801A (en) * | 2011-06-03 | 2012-01-11 | 南开大学 | Metabolic labeling method of antibody and application of antibody in fluorescence detection |
| CN104039977A (en) * | 2012-01-18 | 2014-09-10 | 国家科学研究中心 | A method for specifically labeling living bacteria |
| CN105683389A (en) * | 2013-10-30 | 2016-06-15 | 国家科学研究中心 | Method for labeling specifically living bacteria comprising the use of modified monosaccharide compounds |
| CN104749369A (en) * | 2013-12-31 | 2015-07-01 | 中国科学院深圳先进技术研究院 | Fluorescent labeling method for living organisms having cell membrane structures |
| CN104459125A (en) * | 2014-12-10 | 2015-03-25 | 中国科学院苏州生物医学工程技术研究所 | Method for rapidly detecting gram negative and positive bacteria |
Non-Patent Citations (4)
| Title |
|---|
| ELIANA SAXON ET AL.: "Cell Surface Engineering by a ModiÞed Staudinger Reaction", 《SCIENCE》 * |
| KELLY N. CHUH ET AL.: "Changes in Metabolic Chemical Reporter Structure Yield a Selective Probe of O‑GlcNAc Modification", 《J. AM. CHEM. SOC.》 * |
| 杨麦云等: "生物正交标记反应研究进展", 《化学学报》 * |
| 王静华等: "细菌细胞壁肽聚糖的研究进展", 《中国兽药杂志》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021015622A1 (en) * | 2019-07-25 | 2021-01-28 | Synaffix B.V. | Synthesis of 6-azido-6-deoxy-2-n-acetyl-hexosamine-nucleoside diphosphate |
| NL2023572B1 (en) * | 2019-07-25 | 2021-02-10 | Synaffix Bv | Synthesis of 6-azido-6-deoxy-2-N-acetyl-hexosamine-nucleoside diphosphate |
| CN114423773A (en) * | 2019-07-25 | 2022-04-29 | 西纳福克斯股份有限公司 | Synthesis of 6-azido-6-deoxy-2-N-acetyl-hexosamine-nucleoside diphosphate |
| CN111733102A (en) * | 2020-06-30 | 2020-10-02 | 东南大学 | Gram-positive bacterium surface modification method based on tyrosinase catalysis and application thereof |
| CN111733102B (en) * | 2020-06-30 | 2023-08-18 | 东南大学 | Tyrosinase catalysis-based gram-positive bacterium surface modification method and application thereof |
| CN114790435A (en) * | 2021-01-25 | 2022-07-26 | 山东大学 | Bacillus subtilis engineering strain for synthesizing click-reaction glycosaminoglycan skeleton and construction method and application thereof |
| CN114790435B (en) * | 2021-01-25 | 2023-11-17 | 山东大学 | Bacillus subtilis engineering strain for synthesizing clickable glycosaminoglycan skeleton and construction method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108865918B (en) | 2022-01-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Amann et al. | Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes | |
| Atta | Biochemical studies on antibiotic production from Streptomyces sp.: Taxonomy, fermentation, isolation and biological properties | |
| US9670476B2 (en) | Enzyme substrate comprising a functional dye and associated technology and methods | |
| EP1937850B1 (en) | Methods and compositions for labeling nucleic acids | |
| US9228240B2 (en) | Methods for detecting and quantifying viable bacterial endo-spores | |
| CN108865918A (en) | A method of the gram-positive bacterium of label survival and its cell wall | |
| Franzini et al. | Improved templated fluorogenic probes enhance the analysis of closely related pathogenic bacteria by microscopy and flow cytometry | |
| US20200087703A1 (en) | Metabolic labeling of bacterial teichoic acids cell wall | |
| CN107739749B (en) | Method for screening paralytic shellfish poison producing strain by DNA probe-colony in situ hybridization technology | |
| EP3380841B1 (en) | Autonomous sensing molecules (asm) | |
| Kakinuma et al. | An expeditious chemo-enzymatic route from glucose to catechol by the use of 2-deoxy-scyllo-inosose synthase | |
| Schmidt et al. | Evaluation of tyramide solutions for an improved detection and enumeration of single microbial cells in soil by CARD-FISH | |
| Schouten et al. | Fluorescent reagents for in vitro studies of lipid-linked steps of bacterial peptidoglycan biosynthesis: derivatives of UDPMurNAc-pentapeptide containing D-cysteine at position 4 or 5 | |
| FR2921064A1 (en) | OLIGONUCLEOTIDES, USE, DETECTION METHOD AND KIT FOR DIAGNOSING THE PRESENCE OF THE CHIKUNGUNYA VIRUS E1 GENE | |
| Filion et al. | Permeabilization and hybridization protocols for rapid detection of Bacillus spores using fluorescence in situ hybridization | |
| CN104911187B (en) | Mycobacterium tuberculosis type strain H37DNA aptamer of Rv and preparation method thereof | |
| CN110596383A (en) | Fluorescent dyeing-based rapid magnetic separation and identification method and kit for general pathogenic microorganisms | |
| CN107383927B (en) | A kind of nitrine indoles dimethime Molecule of Cyanine Dyes and application | |
| CN107937490A (en) | The method and kit of a kind of ELISA test strip phytopathogen of asymmetric pcr | |
| Lai et al. | Exploitation of stem-loop DNA as a dual-input gene sensing platform: extension to subtyping of influenza A viruses | |
| CN106432369A (en) | Synthesis method of glucoside based on indoxyl derivative and 2-(benzothiazol-2'-yl)phenol derivative | |
| Yang et al. | Engineered production and evaluation of 6′-deoxy-tallysomycin H-1 revealing new insights into the structure–activity relationship of the anticancer drug bleomycin | |
| Bago et al. | Fluorescently-primed in situ PCR in arbuscular mycorrhizas | |
| Englert et al. | Aptamer-based proximity labeling guides covalent RNA modification | |
| JP2000184894A (en) | One pair of nucleic acid probes and measurement of amount of specific group or specific microorganism existing in composite microorganism system or symbiofic microorganism system with the probes |
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 | ||
| 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: 20220107 |