CN114875095A - 一种丙氨酰美登醇及其合成方法和应用 - Google Patents
一种丙氨酰美登醇及其合成方法和应用 Download PDFInfo
- Publication number
- CN114875095A CN114875095A CN202210598105.6A CN202210598105A CN114875095A CN 114875095 A CN114875095 A CN 114875095A CN 202210598105 A CN202210598105 A CN 202210598105A CN 114875095 A CN114875095 A CN 114875095A
- Authority
- CN
- China
- Prior art keywords
- maytansinol
- alanyl
- gene
- nrps
- astc
- 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
- QWPXBEHQFHACTK-UHFFFAOYSA-N Maytansinol Natural products CN1C(=O)CC(O)C2(C)OC2C(C)C(OC(=O)N2)CC2(O)C(OC)C=CC=C(C)CC2=CC(OC)=C(Cl)C1=C2 QWPXBEHQFHACTK-UHFFFAOYSA-N 0.000 title claims abstract description 38
- -1 Alanyl maytansinol Chemical compound 0.000 title claims abstract description 37
- 238000010189 synthetic method Methods 0.000 title description 3
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 43
- 239000012634 fragment Substances 0.000 claims abstract description 29
- 101100217185 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) aruC gene Proteins 0.000 claims abstract description 20
- 101150024707 astC gene Proteins 0.000 claims abstract description 20
- 229920001184 polypeptide Polymers 0.000 claims abstract description 18
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 18
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 18
- 210000003705 ribosome Anatomy 0.000 claims abstract description 18
- 239000003814 drug Substances 0.000 claims abstract description 11
- 239000013604 expression vector Substances 0.000 claims abstract description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 69
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 33
- 238000000034 method Methods 0.000 claims description 16
- 239000007787 solid Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 241000894006 Bacteria Species 0.000 claims description 10
- 239000000401 methanolic extract Substances 0.000 claims description 10
- 239000013600 plasmid vector Substances 0.000 claims description 10
- 230000002829 reductive effect Effects 0.000 claims description 10
- 238000010898 silica gel chromatography Methods 0.000 claims description 10
- 230000002194 synthesizing effect Effects 0.000 claims description 10
- 238000012408 PCR amplification Methods 0.000 claims description 9
- 230000002441 reversible effect Effects 0.000 claims description 9
- 239000002609 medium Substances 0.000 claims description 8
- 238000012546 transfer Methods 0.000 claims description 8
- 230000000259 anti-tumor effect Effects 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 239000000287 crude extract Substances 0.000 claims description 6
- 239000000284 extract Substances 0.000 claims description 6
- 238000000855 fermentation Methods 0.000 claims description 6
- 230000004151 fermentation Effects 0.000 claims description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000000499 gel Substances 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 238000004809 thin layer chromatography Methods 0.000 claims description 5
- 241000186046 Actinomyces Species 0.000 claims description 4
- 241000588722 Escherichia Species 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 4
- 239000000741 silica gel Substances 0.000 claims description 4
- 229910002027 silica gel Inorganic materials 0.000 claims description 4
- 230000001131 transforming effect Effects 0.000 claims description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 3
- 229920001817 Agar Polymers 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- ONBIUAZBGHXJDM-UHFFFAOYSA-J bismuth;potassium;tetraiodide Chemical compound [K+].[I-].[I-].[I-].[I-].[Bi+3] ONBIUAZBGHXJDM-UHFFFAOYSA-J 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 238000004440 column chromatography Methods 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 235000019253 formic acid Nutrition 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000003480 eluent Substances 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 abstract description 13
- 238000004519 manufacturing process Methods 0.000 abstract description 10
- 229940079593 drug Drugs 0.000 abstract description 9
- 229940127121 immunoconjugate Drugs 0.000 abstract description 9
- 238000011160 research Methods 0.000 abstract description 7
- 238000001308 synthesis method Methods 0.000 abstract description 4
- QWPXBEHQFHACTK-KZVYIGENSA-N (10e,12e)-86-chloro-12,14,4-trihydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-15,16-dihydro-14h-7-aza-1(6,4)-oxazina-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-6-one Chemical compound CN1C(=O)CC(O)C2(C)OC2C(C)C(OC(=O)N2)CC2(O)C(OC)\C=C\C=C(C)\CC2=CC(OC)=C(Cl)C1=C2 QWPXBEHQFHACTK-KZVYIGENSA-N 0.000 abstract description 3
- 241000123663 Actinosynnema Species 0.000 abstract description 3
- 230000014509 gene expression Effects 0.000 abstract description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 abstract description 3
- 230000001737 promoting effect Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 23
- 239000000243 solution Substances 0.000 description 9
- 206010006187 Breast cancer Diseases 0.000 description 8
- 208000026310 Breast neoplasm Diseases 0.000 description 8
- 206010008342 Cervix carcinoma Diseases 0.000 description 7
- 206010009944 Colon cancer Diseases 0.000 description 7
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 7
- 201000010881 cervical cancer Diseases 0.000 description 7
- 208000029742 colonic neoplasm Diseases 0.000 description 7
- IOOMXAQUNPWDLL-UHFFFAOYSA-N 2-[6-(diethylamino)-3-(diethyliminiumyl)-3h-xanthen-9-yl]-5-sulfobenzene-1-sulfonate Chemical compound C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=C(S(O)(=O)=O)C=C1S([O-])(=O)=O IOOMXAQUNPWDLL-UHFFFAOYSA-N 0.000 description 6
- 229940125904 compound 1 Drugs 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 125000001980 alanyl group Chemical group 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 229960001612 trastuzumab emtansine Drugs 0.000 description 5
- 238000000137 annealing Methods 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 230000001472 cytotoxic effect Effects 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 239000002246 antineoplastic agent Substances 0.000 description 3
- 229940041181 antineoplastic drug Drugs 0.000 description 3
- 230000004186 co-expression Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 2
- 239000000611 antibody drug conjugate Substances 0.000 description 2
- 229940049595 antibody-drug conjugate Drugs 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 108091008053 gene clusters Proteins 0.000 description 2
- 230000005918 in vitro anti-tumor Effects 0.000 description 2
- 125000005647 linker group Chemical group 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 208000017891 HER2 positive breast carcinoma Diseases 0.000 description 1
- 238000010268 HPLC based assay Methods 0.000 description 1
- 101710116034 Immunity protein Proteins 0.000 description 1
- 240000001427 Mallotus nudiflorus Species 0.000 description 1
- 102000029749 Microtubule Human genes 0.000 description 1
- 108091022875 Microtubule Proteins 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 241000187180 Streptomyces sp. Species 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102000004243 Tubulin Human genes 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- PVNFMCBFDPTNQI-UIBOPQHZSA-N [(1S,2R,5S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl] acetate [(1S,2R,5S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl] 3-methylbutanoate [(1S,2R,5S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl] 2-methylpropanoate [(1S,2R,5S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl] propanoate Chemical compound CO[C@@H]1\C=C\C=C(C)\Cc2cc(OC)c(Cl)c(c2)N(C)C(=O)C[C@H](OC(C)=O)[C@]2(C)OC2[C@H](C)[C@@H]2C[C@@]1(O)NC(=O)O2.CCC(=O)O[C@H]1CC(=O)N(C)c2cc(C\C(C)=C\C=C\[C@@H](OC)[C@@]3(O)C[C@H](OC(=O)N3)[C@@H](C)C3O[C@@]13C)cc(OC)c2Cl.CO[C@@H]1\C=C\C=C(C)\Cc2cc(OC)c(Cl)c(c2)N(C)C(=O)C[C@H](OC(=O)C(C)C)[C@]2(C)OC2[C@H](C)[C@@H]2C[C@@]1(O)NC(=O)O2.CO[C@@H]1\C=C\C=C(C)\Cc2cc(OC)c(Cl)c(c2)N(C)C(=O)C[C@H](OC(=O)CC(C)C)[C@]2(C)OC2[C@H](C)[C@@H]2C[C@@]1(O)NC(=O)O2 PVNFMCBFDPTNQI-UIBOPQHZSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 125000004202 aminomethyl group Chemical group [H]N([H])C([H])([H])* 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 229930190775 ansacarbamitocin Natural products 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 125000004185 ester group Chemical group 0.000 description 1
- 150000002148 esters Chemical group 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 1
- 238000003929 heteronuclear multiple quantum coherence Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 150000003951 lactams Chemical class 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 1
- 210000004688 microtubule Anatomy 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 239000006225 natural substrate Substances 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 238000005556 structure-activity relationship Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5386—1,4-Oxazines, e.g. morpholine spiro-condensed or forming part of bridged ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Biophysics (AREA)
- Epidemiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及一种丙氨酰美登醇及其合成方法和应用。本发明截取了基因astC中编码A‑T结构域的DNA片段和基因contig_44136中编码TE结构域的DNA片段,插入到整合型表达载体pSBT11上形成杂合基因nrps,构建表达载体pSBT11‑nrps,实现了在珍贵橙色束丝放线菌突变株中异源表达非核糖体多肽合酶基因,进而制备丙氨酰美登醇的目的,并且还通过TE结构域替换,使杂合非核糖体多肽合酶可以更高效地识别细菌美登木素作为氨基受体,使得丙氨酰美登醇的产量达到了0.5mg/L,有效克服了现有技术中存在的产量低的问题,降低了成本,可以用于并促进美登木素抗体偶联物药物的研究、生产和临床应用。
Description
技术领域
本发明涉及一种丙氨酰美登醇及其合成方法和应用,属于天然药物化学与医药应用技术领域。
背景技术
美登木素属于安莎类大环内酰胺,根据其来源可分为植物美登木素和细菌美登木素两大类。两类美登木素具有相同的骨架和相似的后修饰基团,都有极强的抗菌和抗肿瘤活性。研究发现美登木素衍生物可以结合微管蛋白β亚基,阻止微管束聚集形成,破坏有丝分裂过程,从而抑制肿瘤细胞生长。由于存在神经毒性,美登木素衍生物不能直接用于临床,但可以作为“弹头”与特异免疫蛋白偶联制备成抗体药物偶联物,特异性识别肿瘤细胞表面的抗原表位,避免对正常细胞的杀伤,相比于一般化疗药物具有更优越的安全性和耐受性。2013年,FDA 批准了美登木素抗体药物偶联物ado-trastuzumab emtansine(T-DM1)的上市,应用于人表皮生长因子受体2(HER2)阳性乳腺癌的临床治疗。目前,与T-DM1相关的临床研究均证实其在 HER2阳性乳腺癌治疗中具有卓越的疗效。
丙氨酰美登醇衍生物是T-DM1“弹头”的重要中间体,是以细菌美登木素为原料通过化学合成制备的。由于细菌美登木素原始结构中并没有合适的偶联的位点,化学方法需要通过还原反应脱去细菌美登木素C-3位酯基,然后再选择性丙氨酰化,不但成本高、产率低,还有不易分离除去的副产物,造成昂贵原料的浪费,是导致T-DM1价格高昂的重要因素之一。
非核糖体多肽合酶AstC由三个结构域(adenylation-thiolation-thioesterase,A-T-TE)组成,可以使用细菌美登木素为氨基受体,在其C-3位羟基上加入丙氨酰基,然后通过异源表达非核糖体多肽合酶基因astC可以产生C-3位为丙氨酰基的化合物,但通过非核糖体多肽合酶AstC制备丙氨酰美登醇的产量很低,导致合成美登木素抗体偶联物的中间产物成本很高,严重阻碍美登木素抗体偶联物药物的研究、生产和临床应用。
研究表明AstC的TE结构域负责氨基受体的选择,而细菌美登木素不是AstC的天然底物,因此寻找可以高效识别细菌美登木素的非核糖体多肽合酶或TE结构域是丙氨酰美登醇高产的关键。
发明内容
对美登木素衍生物抗肿瘤构效关系的研究发现,C-3位酯基侧链对其抗肿瘤活性起关键作用(Chem Pharm Bull 2004,52,1-26),用于制备美登木素抗体偶联物的是C-3位为丙氨酰基的美登醇衍生物。虽然异源表达非核糖体多肽合酶基因astC可以产生C-3位为丙氨酰基的化合物,但产量极低。根据目前的假说,植物内生菌可能是植物美登木素的真正生产者。对产美登木素植物Mallotus nudiflorus L的茎皮内生菌宏基因组进行分析,发现了可能负责美登木素丙氨酰化的非核糖体多肽合酶基因片段contig_44136,该片段编码T-TE两个结构域,缺少 A结构域,无法独立参与丙氨酰化反应。
针对现有技术的不足,本发明提供一种丙氨酰美登醇及其合成方法和应用。本发明的目标在于通过生物合成高效制备T-DM1“弹头”的重要中间体丙氨酰美登醇,将极大加快美登木素抗体偶联物药物的研发进程,对于发现广谱高效且具有自主知识产权的新型抗肿瘤药物具有重要意义。
本发明的技术方案如下:
一种丙氨酰美登醇,其化学结构如下所示:
上述丙氨酰美登醇的合成方法,包括以下步骤:
(1)将能够表达非核糖体多肽合酶的基因工程菌HGF052+pJTU824-asm18+pSBT11-nrps 在28~30℃下,使用YMG固体培养基发酵培养10~14天,得到固体培养物;
(2)将固体培养物切成1cm见方的小块,在20~30℃下用体积比为80:15:5的乙酸乙酯/ 甲醇/甲酸浸泡提取三次,合并提取液,减压及35~40℃的温度下浓缩至干,得粗提物;
(3)将粗提物溶于水中,使用乙酸乙酯萃取,乙酸乙酯相减压及35~40℃的温度下浓缩至干,得EA提取物;
(4)将EA提取物溶于甲醇中,再经石油醚多次萃取,甲醇相减压及35~40℃的温度下浓缩至干,得甲醇提取物;
(5)将甲醇提取物依次经反相硅胶柱层析分离,凝胶柱层析分离,薄层层析和正相硅胶柱层析分离,然后将组分相同的洗脱液进行合并,得到丙氨酰美登醇。
根据本发明优选的,步骤(1)中,所述基因工程菌HGF052+pJTU824-asm18+pSBT11-nrps 的构建方法,包括步骤如下:
(a)以非核糖体多肽合酶基因astC为模板进行PCR扩增,扩增得到基因astC中编码A-T结构域的DNA片段,PCR引物序列如下:
astC-F:5′-AAAGGAGGCGGACATATGGAGACGAACATGCTGGTGCAGG-3′,
astC-R:5′-CGACCCACGGAGGTCGAAGAAGCTGTCGCGCGGACCGACC-3′;
(b)以人工合成基因contig_44136为模板进行PCR扩增,扩增得到基因contig_44136 中编码TE结构域的DNA片段,PCR引物序列如下:
44136-F:5′-CGCGACAGCTTCTTCGACCTCCGTGGGTCGGACGTGCTCG-3′,
44136-R:5′-GACATGATTACGAATTCAGGGCCGGGTCGGGTCCGTCCCG-3′;
(c)将步骤(1)中所述编码A-T结构域的DNA片段和步骤(2)中所述编码TE结构域的DNA片段顺序插入到整合型表达载体pSBT11上的Ned I和EcoR I限制性酶切位点之间,形成杂合基因nrps,所述杂合基因nrps位于ermE*启动子的下游并受其调控,得到质粒载体pSBT11-nrps;
(d)将质粒载体pSBT11-nrps转化大肠杆菌ET12567/pUZ8002,获得大肠杆菌-放线菌接合转移供体菌ET12567/pUZ8002/pSBT11-nrps;
(e)将供体菌ET12567/pUZ8002/pSBT11-nrps与珍贵橙色束丝放线菌突变株HGF052+pJTU824-asm18的菌丝体进行接合转移,得到基因工程菌 HGF052+pJTU824-asm18+pSBT11-nrps。
进一步优选的,步骤(a)中,所述非核糖体多肽合酶基因astC基因库登记号为KF813023.1,所述基因astC中编码A-T结构域的DNA片段的序列如SEQ ID NO.1所示。
进一步优选的,步骤(b)中,所述人工合成基因contig_44136的序列如SEQ IDNO.4 所示;所述基因contig_44136中编码TE结构域的DNA片段的序列如SEQ ID NO.2所示。
进一步优选的,步骤(c)中,所述整合型表达载体pSBT11来自文献Li,X.,et al.(2019). "Identification of the bacterial maytansinoid gene cluster ascprovides insights into the post-PKS modifications of ansacarbamitocinbiosynthesis."Organic Letters 21(15):5823-5826。
进一步优选的,步骤(c)中,所述杂合基因nrps序列如SEQ ID NO.3所示。
进一步优选的,步骤(e)中,所述HGF052+pJTU824-asm18菌株来自文献ApplMicrobiol Biotechnol 2016,100,2641-2649。
根据本发明优选的,步骤(1)中,所述发酵培养条件为在28℃下培养10天,所述YMG固体培养基成分以质量百分比计为:0.4%葡萄糖、1%麦芽提取物、0.4%酵母提取物、2%琼脂粉和96.2%蒸馏水。
根据本发明优选的,步骤(4)中,所述提取采用的甲醇为90~95%甲醇。
根据本发明优选的,步骤(5)中,所述反相硅胶柱填料为C-18,凝胶柱型号为Sephadex LH-20,正相硅胶柱填料为200~300目。
根据本发明优选的,步骤(5)中,所述对甲醇提取物进行分离的具体步骤为:
甲醇提取物首先经反相硅胶柱层析分离,分别用水、40%、60%、80%、100%甲醇依次洗脱,每个组分洗脱1L,200~250mL/份接收,TLC检测,用CH2Cl2:MeOH=15:1(v/v)展开,用浓硫酸和碘化铋钾显色,合并60%洗脱组分;继续用凝胶柱层析分离,甲醇洗脱,3~5mL/管,合并33~38管;继续正相硅胶柱层析分离,最后用体积比为100:1、80:1、50:1的CH2Cl2/MeOH溶液洗脱,合并80:1洗脱成分,得到丙氨酰美登醇。
经药理试验研究表明,本发明提供的丙氨酰美登醇对人宫颈癌细胞(HeLa)、人结肠癌细胞(HCT116)和人乳腺癌细胞(MDA-MB-231)显示了明显的细胞毒活性,IC50值分别为6.3、3.1和5.2nM,本发明还提供丙氨酰美登醇的制药用途,可用于制备抗肿瘤药物。优选的,所述的肿瘤为宫颈癌、结肠癌或乳腺癌。
一种抗肿瘤的药物组合物,包括本发明的丙氨酰美登醇和一种或多种药学上可接受载体或赋形剂。
本发明未详尽之处,均可采用现有技术。
本发明的有益效果在于:
1、经体外抗肿瘤活性试验表明,本发明提供的丙氨酰美登醇对人宫颈癌细胞(HeLa)、人结肠癌细胞(HCT116)和人乳腺癌细胞(MDA-MB-231)显示了明显的细胞毒活性,IC50 值分别为6.3、3.1和5.2nM,因此可用于制备抗肿瘤药物,可与不同的抗体和连接子组成抗体偶联物。
2、本发明截取了基因astC中编码A-T结构域的DNA片段和基因contig_44136中编码 TE结构域的DNA片段,并将这两段基因插入到整合型表达载体pSBT11上形成杂合基因nrps,构建了基因nrps的整合型表达载体pSBT11-nrps,实现了在珍贵橙色束丝放线菌突变株中异源表达非核糖体多肽合酶基因,进而制备丙氨酰美登醇的目的,并且还通过TE结构域替换,使杂合非核糖体多肽合酶可以更高效地识别细菌美登木素作为氨基受体,使得丙氨酰美登醇的产量达到了0.5mg/L,有效克服了现有技术中存在的产量低的问题,降低了成本,可以用于并促进美登木素抗体偶联物药物的研究、生产和临床应用。
3、本发明通过生物合成方法得到的肿瘤抑制活性的丙氨酰美登醇,避免了传统的化学合成方法带来的原料浪费和难以去除的副产物。
附图说明
图1为HPLC检测实施例1基因工程菌和对比例共表达菌株的培养物中丙氨酰美登醇产量。
图中:A为基因工程菌为HGF052+pJTU824-asm18+pSBT11-nrps;B为共表达菌株HGF052+pJTU824-asm18+pSBT11-astC-contig44136;1为丙氨酰美登醇。
具体实施方式
下面结合附图和具体实施例对本发明作进一步详细说明,但不应理解为对本发明的限制。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
安丝菌素AP-3*,购自MCE公司(MedChemExpress)。
人宫颈癌细胞(HeLa)、人结肠癌细胞(HCT116)和人乳腺癌细胞(MDA-MB-231),中科院上海细胞库有售。
整合型表达载体pSBT11来自文献Li,X.,et al.(2019)."Identification of thebacterial maytansinoid gene cluster asc provides insights into the post-PKSmodifications of ansacarbamitocin biosynthesis."Organic Letters 21(15):5823-5826。
HGF052+pJTU824-asm18菌株来自文献Appl Microbiol Biotechnol 2016,100,2641-2649。
在以下实施例中所述化合物1的化学结构式如下所示(结构式中的阿拉伯数字是化学结构中碳原子的标位):
实施例1构建基因工程菌
一种基因工程菌HGF052+pJTU824-asm18+pSBT11-nrps的构建方法,包括步骤如下:
(a)以非核糖体多肽合酶基因astC为模板进行PCR扩增,扩增得到基因astC中编码A-T结构域的DNA片段,PCR引物序列如下:
astC-F:5′-AAAGGAGGCGGACATATGGAGACGAACATGCTGGTGCAGG-3′,
astC-R:5′-CGACCCACGGAGGTCGAAGAAGCTGTCGCGCGGACCGACC-3′;
PCR体系为:2×PrimeSTAR Max Premix,25μL;Forward primer(10μM),1μL;Reverse primer(10μM),1μL;Template,50ng;补足ddH2O至50μL;
PCR扩增程序:变性,98℃10sec;退火,55℃30sec;延伸,72℃1min(30~35个循环);终止延伸,72℃10min;最后4℃保温;
所述非核糖体多肽合酶基因astC基因库登记号为KF813023.1,所述基因astC中编码A-T 结构域的DNA片段的序列如SEQ ID NO.1所示;
(b)由北京擎科生物科技有限公司合成基因contig_44136,然后以人工合成基因contig_44136为模板进行PCR扩增,扩增得到基因contig_44136中编码TE结构域的DNA片段,PCR引物序列如下:
44136-F:5′-CGCGACAGCTTCTTCGACCTCCGTGGGTCGGACGTGCTCG-3′,
44136-R:5′-GACATGATTACGAATTCAGGGCCGGGTCGGGTCCGTCCCG-3′;
PCR体系为:2×PrimeSTAR Max Premix,25μL;Forward primer(10μM),1μL;Reverse primer(10μM),1μL;Template,50ng;补足ddH2O至50μL;
PCR扩增程序:变性,98℃10sec;退火,55℃30sec;延伸,72℃1min(30~35个循环);终止延伸,72℃10min;最后4℃保温;
所述人工合成基因contig_44136的序列如SEQ ID NO.4所示;所述基因contig_44136中编码TE结构域的DNA片段的序列如SEQ ID NO.2所示。
(c)将步骤(1)中所述编码A-T结构域的DNA片段和步骤(2)中所述编码TE结构域的DNA片段顺序插入到整合型表达载体pSBT11上的Ned I和EcoR I限制性酶切位点之间,形成杂合基因nrps,所述杂合基因nrps位于ermE*启动子的下游并受其调控,得到质粒载体pSBT11-nrps;所述杂合基因nrps序列如SEQ ID NO.3所示;
(d)将质粒载体pSBT11-nrps转化大肠杆菌ET12567/pUZ8002,获得大肠杆菌-放线菌接合转移供体菌ET12567/pUZ8002/pSBT11-nrps;
(e)将供体菌ET12567/pUZ8002/pSBT11-nrps与珍贵橙色束丝放线菌突变株HGF052+pJTU824-asm18的菌丝体进行接合转移,得到基因工程菌 HGF052+pJTU824-asm18+pSBT11-nrps。
实施例2化合物1的制备
一种使用基因工程菌制备化合物1的方法,包括以下步骤:
(1)将实施例1所述的基因工程菌HGF052+pJTU824-asm18+pSBT11-nrps在28℃下,使用15L的YMG固体培养基发酵培养10天,得到固体培养物;所述YMG固体培养基成分以质量百分比计为:0.4%葡萄糖、1%麦芽提取物、0.4%酵母提取物、2%琼脂粉和蒸馏水96.2%;
(2)将固体培养物切成1cm见方的小块,在25℃下用体积比为80:15:5的乙酸乙酯/甲醇/甲酸浸泡提取三次,合并提取液,减压及38℃的温度下浓缩至干,得粗提物;
(3)将粗提物溶于水中,使用乙酸乙酯萃取,乙酸乙酯相减压及38℃的温度下浓缩至干,得EA提取物;
(4)将EA提取物溶于甲醇中,再经石油醚多次萃取,甲醇相减压及35~40℃的温度下浓缩至干,得甲醇提取物;
(5)将甲醇提取物首先经反相硅胶柱层析(RP-18,200g柱)分离,然后再分别用水、40%、60%、80%、100%甲醇依次洗脱,每个组分洗脱1L,250mL/份接收,TLC检测,用CH2Cl2:MeOH=15:1(v/v)展开,用浓硫酸和碘化铋钾显色,合并60%洗脱组分;继续用凝胶(Sephadex LH-20)柱层析分离,甲醇洗脱,5mL/管,合并33~38管,继续用正相硅胶柱层析分离,最后用体积比为100:1、80:1、50:1的CH2Cl2/MeOH溶液洗脱,合并80:1洗脱成分,得到的化合物1。
实施例3丙氨酰美登醇的鉴定
电喷雾质谱法(ESI-MS)测得实施例2所得化合物1的准分子离子峰为m/z 636.27[M+ H]+。1H和13C NMR显示,化合物1共含有31个碳原子(表1),包括4个甲基,2个甲氧基,1个氮甲基,3个亚甲基,11个次甲基和10个季碳。根据HMQC和HMBC的信号,确定了该化合物为丙氨酰美登醇。根据C-3位质子与C-1’的远程相关关系,确定了丙氨酰基的取代位置是在C-3位的O上,对全部的NMR光谱数据进行指定,确定为一新化合物。
表1、化合物1的核磁共振数据
实施例4丙氨酰美登醇的体外抗肿瘤活性试验
采用硫酰罗丹明B(sulforhodamine B,SRB)蛋白染色法测定细胞生长抑制率。
具体方法如下:
1)将人宫颈癌细胞(HeLa)、人结肠癌细胞(HCT116)和人乳腺癌细胞(MDA-MB-231)分别培养至对数生长期,胰酶消化,用新鲜的DMEM培养基调整细胞密度至3~7万个/mL,接种于96孔板中,每孔加入100μL细胞,置于37℃、5.0%CO2饱和湿度的培养箱中培养过夜;
2)用DMEM培养基将丙氨酰美登醇浓度稀释至2倍检测浓度,取100μL稀释液加入96孔板中,继续培养72h;
3)弃除细胞培养基,缓慢加入100μL预冷的10%TCA溶液,4℃放置1h以上;去除TCA固定液,用缓慢流动的水冲洗五次,用吸水纸吸干水分;
4)加入100μL SRB染色液,室温孵育30min。去除SRB染色液,用1%的冰醋酸冲洗五遍,去除未结合的SRB染料;室温干燥,加入100μL Tris溶液(10mM,pH为10.0),过夜溶解SRB染料;
5)使用酶标仪在波长570nm下测定给药孔和空白孔OD值,结果如表2所示。
细胞生长抑制率=(1-用药组平均OD值/对照组孔OD值)×100%。
试验结果的评判与解释:细胞半数生长抑制时的药物浓度IC50按照量效数据进行换算。每个实验重复三次,吸收值差异小于5%,IC50差异小于30%,以IC50≤100nM为有效标准。
由表2可知,本发明提供的丙氨酰美登醇对人宫颈癌细胞(HeLa)、人结肠癌细胞(HCT116)和人乳腺癌细胞(MDA-MB-231)均显示了明显的细胞毒活性,其IC50值分别为 6.3、3.1和5.2nM。
试验结论:通过药理学试验,可以看出本发明提供的丙氨酰美登醇对人宫颈癌细胞 (HeLa)、人结肠癌细胞(HCT116)和人乳腺癌细胞(MDA-MB-231)均显示了明显的细胞毒活性。因此,本发明提供的丙氨酰美登醇可用于制备抗肿瘤药物,可与其他药物制成抗肿瘤药物组合物,还可以与不同的抗体和连接子偶联制成抗体偶联物。
表2、丙氨酰美登醇对3株肿瘤细胞的细胞毒试验结果(IC50,nM)
*为阳性对照药
对比例
一种基因astC和基因contig_44136共表达菌株的构建,包括步骤如下:
1)以珍贵橙色束丝放线菌突变株HGF052的基因组为模板,进行PCR扩增,扩增得到基因astC序列,PCR引物序列如下:
astC-F:5′-AAAGGAGGCGGACATATGGAGACGAACATGCTGGTGCAGG-3′,
astC-R2:5′-GACATGATTACGAATTCAGTCGGCGAGGTGGCCGGAGACG-3′;
PCR体系为:2×PrimeSTAR Max Premix,25μL;Forward primer(10μM),1μL;Reverse primer(10μM),1μL;Template,50ng;补足ddH2O至50μL;
PCR扩增程序:变性,98℃10sec;退火,55℃30sec;延伸,72℃1min(30~35个循环);终止延伸,72℃10min;最后4℃保温;
然后通过Gibson组装将基因astC序列插入到整合型表达载体pSBT11上的Ned I和EcoR I限制性酶切位点之间,得到质粒载体pSBT11-astC;
2)以pSBT11为模板,进行PCR扩增,扩增得到ermE*启动子序列,PCR引物序列如下:
ermE-F:5′-TCGCCGACTGAATTCGTGCACGCGGTCGATCTTGACGGCT-3′,
ermE-R:5′-GCATCTCCTCGGCGGATGTCCGCCTCCTTTGGTCGATATG-3′;
PCR体系为:2×PrimeSTAR Max Premix,25μL;Forward primer(10μM),1μL;Reverse primer(10μM),1μL;Template,50ng;补足ddH2O至50μL;
PCR扩增程序:变性,98℃10sec;退火,55℃30sec;延伸,72℃1min(30~35个循环);终止延伸,72℃10min;最后4℃保温;
3)由北京擎科生物科技有限公司合成基因contig_44136,然后以该序列为模板进行PCR 扩增,得到基因contig_44136的扩增产物,PCR引物序列如下:
44136-F2:5′-AAAGGAGGCGGACATCCGCCGAGGAGATGCTCGCGATCGT-3′,
44136-R2:5′-CTATGACATGATTACGAATTCCATGCGCGGCCCTCGCCGA-3′;
4)通过Gibson组装将ermE*启动子序列和基因contig_44136序列插入到质粒载体pSBT11-astC的EcoR I限制性酶切位点之间,得到质粒载体pSBT11-astC-contig_44136;
5)将质粒载体pSBT11-astC-contig_44136转化大肠杆菌ET12567/pUZ8002,获得大肠杆菌-放线菌接合转移供体菌ET12567/pUZ8002/pSBT11-astC-contig_44136;
6)将供体菌ET12567/pUZ8002/pSBT11-astC-contig_44136与珍贵橙色束丝放线菌突变株 HGF052+pJTU824-asm18的菌丝体进行接合转移,得到共表达菌株HGF052+pJTU824-asm18+ pSBT11-astC-contig_44136。
试验例
按照实施例2所述方法对实施例1基因工程菌HGF052+pJTU824-asm18+pSBT11-nrps和对比例1共表达菌株HGF052+pJTU824-asm18+pSBT11-astC-contig_44136分别进行固体培养,然后对固体培养物进行HPLC检测,结果如图1所示。
HPLC检测使用Agilent公司的HPLC系统(1260),使用ZORBAX RX-C18色谱柱(250mm×4.6mm,5μm)。检测波长为254nm,流速为1mL/min,流动相A为ddH2O,流动相B 为乙腈,洗脱条件见表2。
表2发酵产物HPLC检测方法
由图1可知,以实施例1基因工程菌HGF052+pJTU824-asm18+pSBT11-nrps为生产菌株制备丙氨酰美登醇的产量为7.5mg,以YMG固体培养基计,丙氨酰美登醇的产量为0.5mg/L。而以对比例共表达菌株HGF052+pJTU824-asm18+pSBT11-astC-contig_44136为生产菌株,制备丙氨酰美登醇的产量为1.2mg,以YMG固体培养基计,丙氨酰美登醇的产量为0.08mg/L。即本发明通过截取了基因astC中编码A-T结构域的DNA片段和基因contig_44136中编码TE 结构域的DNA片段形成杂合基因nrps,再表达基因nrps制备丙氨酰美登醇,有效的提高了丙氨酰美登醇的产量,是对比菌株的6.25倍,有效克服了现有技术中存在的产量低的问题,降低了成本,可以用于并促进美登木素抗体偶联物药物的研究、生产和临床应用。
SEQUENCE LISTING
<110> 山东大学
<120> 一种丙氨酰美登醇及其合成方法和应用
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1617
<212> DNA
<213> Streptomyces sp.
<400> 1
atggagacga acatgctggt gcaggcgctg gaggaacggg ccgacaccgt tccgtacctc 60
acggccctcg aatgcgaggg cgaggagctc accttccggg aggcgcacga gcgggccgac 120
cggctcgccc gccacctggt cgcccagggg gtgggacccg accgcgtcgt cgcggtcatg 180
ctgccgcgct ccacggacct cctcgtggcg ctcctcgcgg tgctgaaggc gggcggcgcc 240
tacctcgcgc tcgacccgga acaccccgat gagcgcgtcg cgttccaggt ccgcgacgcc 300
gcccccgtcg tgctgctgac ctcgtcccgc atcgacgccg accgcacccg gctgggcttg 360
cccaccgtcg tcctcgacga cccggcgacc gccgagaccc tggcaggctt gcccgccggg 420
cacctcacgg acgccgagcg gaccgccccg acgggccccg aggacctcgc ctacgtcatc 480
tacacctcgg gctccaccgg cacccccaag ggcgtcgaga tccccgtgcg cgccctgcac 540
aacctcctgg aggcgatgcg ggagcggctg ggcctgggcc ccggcgaccg catgctgtcg 600
gtgaccaccg ccaccttcga catgtcggtg cccgagctgt tcctgccgta ctacaccggc 660
gcccgcgcgg tcatcgcgcc ccgggccacc ggacaggacc cgcaggcgct gggcgacctg 720
atcgtccgcc gggagatcgg caccgcgcag gccacgccca cccactggca catgctgtcc 780
accgtcagcc ccgaggtgct gcgcggcctg cgcatcctca tcggcggcga ggcgctctcg 840
gagaagctgg ccgcgaccct gctcgacctc ggcgctgagg tcgtccagtg gtacgggccc 900
accgagacga ccgtgtggtc caccgtccac ccggtcaccg gccccgagga cgccgctgtc 960
atcggccggc cgctgcgcaa cacccggctg tacgtcctgg acgaggacct cgagcccgtc 1020
gcccccggca ccgagggcga gctcttcgtc gccggcgacg gcgtggcgcg cggctacctc 1080
aaccggcccg agctgaccgc cgagcacttc ctgcccgacc gcgacggcga cgccctgatg 1140
taccgcaccg gcgacgtggt gcggctgcgt cccgacggca acctggagta cgtcggccgc 1200
gccgaccacc aggtcaaact gcacggcttc cgcatggagc tcggcgagat cgaggccgcc 1260
ctggagcggt ccgaggacgt cgaccaggcg gccgcgacgg tccgcgagga ccggccgggc 1320
gaccggcgcc tggtggccta cgtgaccgcc gccgcggacc gcacgcccga cgccgggcag 1380
ctgcgcgact tcgtcgccga ggcgctgccc ctgtacatgg tgccgtccgc cgtggtgacc 1440
ctcggggagt ttccgctcac gcccaacggc aagctcgacc gcaaggccct gccggccccc 1500
gtcgtcaccg cgacggccat ggtcgagacc cacctcactc agtcggagct gctgctcggc 1560
aggctcttcg ccgaggtgct cggcatgggc caggtcggtc cgcgcgacag cttcttc 1617
<210> 2
<211> 894
<212> DNA
<213> 人工序列
<400> 2
gacctccgtg ggtcggacgt gctcgaacac cccaccctgc gggccttcgc gcgccgggtg 60
cgtgtcggca ccgcggcgct gccgagccac cccgacgtcg tgaaggtctc cgaggcccgg 120
gcctcgggca acccggcggt gttctgcttc gccggcgccg gagcgctcgc cctgaccttc 180
ctgccgctgt cgcggtacct gcccgagtac gacgtgtacg cgttccagca gcaggccctc 240
gagcgccgtg gggtcccgga ctggtccgtc acccgcagcg cccgccggta cctcgcgctc 300
atgcggatcg tgcagccgcg cggcccgtac ctgctcgtcg gccactcgct cggcgggctc 360
atcgcgctcg agatcgcacg gctcctcacc gagggcggcg agcgggtcca acacgtcgtc 420
ctgctcgaca cctacctgcc gcggagccgg gcggagcagg cgcgcctcga cttcggccgg 480
ttgcggccgc agcagccgtc gaacgcgacg gtgcgcgtcg tccgcaacgg cttggaccgg 540
ctcgcacgac gcgtcctgcc ggccggggtg ccctacggcg agaaggccgc gaagcggttc 600
cgcgcgtaca ccgccggtgt gctccgcttc ggcgggcaga aggacttcga cgcgatgttc 660
gaccacgccg agatgatcgt gcgccggcac acgccgacgc cgttccacgg ccggtcgacc 720
ttcgtgctcg cggacgacaa cccggacgtc gagcgctggt cgtcggtgct ccgcggggac 780
aaccgcaccg tgcacatcca ggcggagcac acgtcgctcc tgcgcgaacc gcacgtggcg 840
gagctcgcag cggagctgcg caccgccctc gggacggacc cgacccggcc ctga 894
<210> 3
<211> 2511
<212> DNA
<213> 人工序列
<400> 3
atggagacga acatgctggt gcaggcgctg gaggaacggg ccgacaccgt tccgtacctc 60
acggccctcg aatgcgaggg cgaggagctc accttccggg aggcgcacga gcgggccgac 120
cggctcgccc gccacctggt cgcccagggg gtgggacccg accgcgtcgt cgcggtcatg 180
ctgccgcgct ccacggacct cctcgtggcg ctcctcgcgg tgctgaaggc gggcggcgcc 240
tacctcgcgc tcgacccgga acaccccgat gagcgcgtcg cgttccaggt ccgcgacgcc 300
gcccccgtcg tgctgctgac ctcgtcccgc atcgacgccg accgcacccg gctgggcttg 360
cccaccgtcg tcctcgacga cccggcgacc gccgagaccc tggcaggctt gcccgccggg 420
cacctcacgg acgccgagcg gaccgccccg acgggccccg aggacctcgc ctacgtcatc 480
tacacctcgg gctccaccgg cacccccaag ggcgtcgaga tccccgtgcg cgccctgcac 540
aacctcctgg aggcgatgcg ggagcggctg ggcctgggcc ccggcgaccg catgctgtcg 600
gtgaccaccg ccaccttcga catgtcggtg cccgagctgt tcctgccgta ctacaccggc 660
gcccgcgcgg tcatcgcgcc ccgggccacc ggacaggacc cgcaggcgct gggcgacctg 720
atcgtccgcc gggagatcgg caccgcgcag gccacgccca cccactggca catgctgtcc 780
accgtcagcc ccgaggtgct gcgcggcctg cgcatcctca tcggcggcga ggcgctctcg 840
gagaagctgg ccgcgaccct gctcgacctc ggcgctgagg tcgtccagtg gtacgggccc 900
accgagacga ccgtgtggtc caccgtccac ccggtcaccg gccccgagga cgccgctgtc 960
atcggccggc cgctgcgcaa cacccggctg tacgtcctgg acgaggacct cgagcccgtc 1020
gcccccggca ccgagggcga gctcttcgtc gccggcgacg gcgtggcgcg cggctacctc 1080
aaccggcccg agctgaccgc cgagcacttc ctgcccgacc gcgacggcga cgccctgatg 1140
taccgcaccg gcgacgtggt gcggctgcgt cccgacggca acctggagta cgtcggccgc 1200
gccgaccacc aggtcaaact gcacggcttc cgcatggagc tcggcgagat cgaggccgcc 1260
ctggagcggt ccgaggacgt cgaccaggcg gccgcgacgg tccgcgagga ccggccgggc 1320
gaccggcgcc tggtggccta cgtgaccgcc gccgcggacc gcacgcccga cgccgggcag 1380
ctgcgcgact tcgtcgccga ggcgctgccc ctgtacatgg tgccgtccgc cgtggtgacc 1440
ctcggggagt ttccgctcac gcccaacggc aagctcgacc gcaaggccct gccggccccc 1500
gtcgtcaccg cgacggccat ggtcgagacc cacctcactc agtcggagct gctgctcggc 1560
aggctcttcg ccgaggtgct cggcatgggc caggtcggtc cgcgcgacag cttcttcgac 1620
ctccgtgggt cggacgtgct cgaacacccc accctgcggg ccttcgcgcg ccgggtgcgt 1680
gtcggcaccg cggcgctgcc gagccacccc gacgtcgtga aggtctccga ggcccgggcc 1740
tcgggcaacc cggcggtgtt ctgcttcgcc ggcgccggag cgctcgccct gaccttcctg 1800
ccgctgtcgc ggtacctgcc cgagtacgac gtgtacgcgt tccagcagca ggccctcgag 1860
cgccgtgggg tcccggactg gtccgtcacc cgcagcgccc gccggtacct cgcgctcatg 1920
cggatcgtgc agccgcgcgg cccgtacctg ctcgtcggcc actcgctcgg cgggctcatc 1980
gcgctcgaga tcgcacggct cctcaccgag ggcggcgagc gggtccaaca cgtcgtcctg 2040
ctcgacacct acctgccgcg gagccgggcg gagcaggcgc gcctcgactt cggccggttg 2100
cggccgcagc agccgtcgaa cgcgacggtg cgcgtcgtcc gcaacggctt ggaccggctc 2160
gcacgacgcg tcctgccggc cggggtgccc tacggcgaga aggccgcgaa gcggttccgc 2220
gcgtacaccg ccggtgtgct ccgcttcggc gggcagaagg acttcgacgc gatgttcgac 2280
cacgccgaga tgatcgtgcg ccggcacacg ccgacgccgt tccacggccg gtcgaccttc 2340
gtgctcgcgg acgacaaccc ggacgtcgag cgctggtcgt cggtgctccg cggggacaac 2400
cgcaccgtgc acatccaggc ggagcacacg tcgctcctgc gcgaaccgca cgtggcggag 2460
ctcgcagcgg agctgcgcac cgccctcggg acggacccga cccggccctg a 2511
<210> 4
<211> 1081
<212> DNA
<213> 人工序列
<400> 4
ccgccgagga gatgctcgcg atcgtgcacc agcggctcgg tgtcgacctc cgtgggtcgg 60
acgtgctcga acaccccacc ctgcgggcct tcgcgcgccg ggtgcgtgtc ggcaccgcgg 120
cgctgccgag ccaccccgac gtcgtgaagg tctccgaggc ccgggcctcg ggcaacccgg 180
cggtgttctg cttcgccggc gccggagcgc tcgccctgac cttcctgccg ctgtcgcggt 240
acctgcccga gtacgacgtg tacgcgttcc agcagcaggc cctcgagcgc cgtggggtcc 300
cggactggtc cgtcacccgc agcgcccgcc ggtacctcgc gctcatgcgg atcgtgcagc 360
cgcgcggccc gtacctgctc gtcggccact cgctcggcgg gctcatcgcg ctcgagatcg 420
cacggctcct caccgagggc ggcgagcggg tccaacacgt cgtcctgctc gacacctacc 480
tgccgcggag ccgggcggag caggcgcgcc tcgacttcgg ccggttgcgg ccgcagcagc 540
cgtcgaacgc gacggtgcgc gtcgtccgca acggcttgga ccggctcgca cgacgcgtcc 600
tgccggccgg ggtgccctac ggcgagaagg ccgcgaagcg gttccgcgcg tacaccgccg 660
gtgtgctccg cttcggcggg cagaaggact tcgacgcgat gttcgaccac gccgagatga 720
tcgtgcgccg gcacacgccg acgccgttcc acggccggtc gaccttcgtg ctcgcggacg 780
acaacccgga cgtcgagcgc tggtcgtcgg tgctccgcgg ggacaaccgc accgtgcaca 840
tccaggcgga gcacacgtcg ctcctgcgcg aaccgcacgt ggcggagctc gcagcggagc 900
tgcgcaccgc cctcgggacg gacccgaccc ggccctgagc cggacctggt ggcggacggg 960
aggctcgggg cgacccccgc cacgagcctc ccgtccgccc gtccgtgcat ccacagccgt 1020
ggcgacacgc ccgggcaggt gctggtagag ttgctgacga cttcggcgag ggccgcgcat 1080
g 1081
Claims (10)
2.如权利要求1所述丙氨酰美登醇的合成方法,其特征在于,包括以下步骤:
(1)将能够表达非核糖体多肽合酶的基因工程菌HGF052+pJTU824-asm18+pSBT11-nrps在28~30℃下,使用YMG固体培养基发酵培养10~14天,得到固体培养物;
(2)将固体培养物切成1cm见方的小块,在20~30℃下用体积比为80:15:5的乙酸乙酯/甲醇/甲酸浸泡提取三次,合并提取液,减压及35~40℃的温度下浓缩至干,得粗提物;
(3)将粗提物溶于水中,使用乙酸乙酯萃取,乙酸乙酯相减压及35~40℃的温度下浓缩至干,得EA提取物;
(4)将EA提取物溶于甲醇中,再经石油醚多次萃取,甲醇相减压及35~40℃的温度下浓缩至干,得甲醇提取物;
(5)将甲醇提取物依次经反相硅胶柱层析分离,凝胶柱层析分离,薄层层析和正相硅胶柱层析分离,然后将组分相同的洗脱液进行合并,得到丙氨酰美登醇。
3.如权利要求2所述丙氨酰美登醇的合成方法,其特征在于,步骤(1)中,所述基因工程菌HGF052+pJTU824-asm18+pSBT11-nrps的构建方法,包括步骤如下:
(a)以非核糖体多肽合酶基因astC为模板进行PCR扩增,扩增得到基因astC中编码A-T结构域的DNA片段,PCR引物序列如下:
astC-F:5′-AAAGGAGGCGGACATATGGAGACGAACATGCTGGTGCAGG-3′,
astC-R:5′-CGACCCACGGAGGTCGAAGAAGCTGTCGCGCGGACCGACC-3′;
(b)以人工合成基因contig_44136为模板进行PCR扩增,扩增得到基因contig_44136中编码TE结构域的DNA片段,PCR引物序列如下:
44136-F:5′-CGCGACAGCTTCTTCGACCTCCGTGGGTCGGACGTGCTCG-3′,
44136-R:5′-GACATGATTACGAATTCAGGGCCGGGTCGGGTCCGTCCCG-3′;
(c)将步骤(1)中所述编码A-T结构域的DNA片段和步骤(2)中所述编码TE结构域的DNA片段顺序插入到整合型表达载体pSBT11上的Ned I和EcoR I限制性酶切位点之间,形成杂合基因nrps,所述杂合基因nrps位于ermE*启动子的下游并受其调控,得到质粒载体pSBT11-nrps;
(d)将质粒载体pSBT11-nrps转化大肠杆菌ET12567/pUZ8002,获得大肠杆菌-放线菌接合转移供体菌ET12567/pUZ8002/pSBT11-nrps;
(e)将供体菌ET12567/pUZ8002/pSBT11-nrps与珍贵橙色束丝放线菌突变株HGF052+pJTU824-asm18的菌丝体进行接合转移,得到基因工程菌HGF052+pJTU824-asm18+pSBT11-nrps。
4.如权利要求3所述丙氨酰美登醇的合成方法,其特征在于,步骤(a)中,所述非核糖体多肽合酶基因astC基因库登记号为KF813023.1,所述基因astC中编码A-T结构域的DNA片段的序列如SEQ ID NO.1所示。
5.如权利要求3所述丙氨酰美登醇的合成方法,其特征在于,步骤(b)中,所述人工合成基因contig_44136的序列如SEQ ID NO.4所示;所述基因contig_44136中编码TE结构域的DNA片段的序列如SEQ ID NO.2所示。
6.如权利要求3所述丙氨酰美登醇的合成方法,其特征在于,步骤(c)中,所述杂合基因nrps序列如SEQ ID NO.3所示。
7.如权利要求2所述丙氨酰美登醇的合成方法,其特征在于,步骤(1)中,所述发酵培养条件为在28℃下培养10天,所述YMG固体培养基成分以质量百分比计为:0.4%葡萄糖、1%麦芽提取物、0.4%酵母提取物、2%琼脂粉和96.2%蒸馏水。
8.如权利要求2所述丙氨酰美登醇的合成方法,其特征在于,步骤(4)中,所述提取采用的甲醇为90~95%甲醇;
步骤(5)中,所述反相硅胶柱填料为C-18,凝胶柱型号为Sephadex LH-20,正相硅胶柱填料为200~300目;所述对甲醇提取物进行分离的具体步骤为:甲醇提取物首先经反相硅胶柱层析分离,分别用水、40%、60%、80%、100%甲醇依次洗脱,每个组分洗脱1L,200~250mL/份接收,TLC检测,用CH2Cl2:MeOH=15:1(v/v)展开,用浓硫酸和碘化铋钾显色,合并60%洗脱组分;继续用凝胶柱层析分离,甲醇洗脱,3~5mL/管,合并33~38管;继续正相硅胶柱层析分离,最后用体积比为100:1、80:1、50:1的CH2Cl2/MeOH溶液洗脱,合并80:1洗脱成分,得到丙氨酰美登醇。
9.权利要求1所述丙氨酰美登醇在制备抗肿瘤药物中的应用。
10.一种抗肿瘤的药物组合物,其特征在于,包括权利要求1所述丙氨酰美登醇和一种或多种药学上可接受载体或赋形剂。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210598105.6A CN114875095B (zh) | 2022-05-30 | 2022-05-30 | 一种丙氨酰美登醇及其合成方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210598105.6A CN114875095B (zh) | 2022-05-30 | 2022-05-30 | 一种丙氨酰美登醇及其合成方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114875095A true CN114875095A (zh) | 2022-08-09 |
CN114875095B CN114875095B (zh) | 2024-05-10 |
Family
ID=82679910
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210598105.6A Active CN114875095B (zh) | 2022-05-30 | 2022-05-30 | 一种丙氨酰美登醇及其合成方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114875095B (zh) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111635418A (zh) * | 2020-06-17 | 2020-09-08 | 山东大学 | 一种美登木素衍生物及其合成方法和应用 |
-
2022
- 2022-05-30 CN CN202210598105.6A patent/CN114875095B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111635418A (zh) * | 2020-06-17 | 2020-09-08 | 山东大学 | 一种美登木素衍生物及其合成方法和应用 |
Non-Patent Citations (2)
Title |
---|
李小曼: "药用功能导向的细菌美登木素合成生物学结构优化", 《中国博士学位论文全文数据库医药卫生科技辑》, no. 2, 15 February 2020 (2020-02-15), pages 079 - 9 * |
李小曼: "药用功能导向的细菌美登木素合成生物学结构优化", 《中国博士学位论文全文数据库医药卫生科技辑》, no. 2, pages 079 - 9 * |
Also Published As
Publication number | Publication date |
---|---|
CN114875095B (zh) | 2024-05-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111635418A (zh) | 一种美登木素衍生物及其合成方法和应用 | |
CN106497827B (zh) | 一种定向生产抗结核活性和抗肿瘤活性化合物的基因工程菌株及其应用 | |
CN108048369B (zh) | 一种产星形孢菌素的海洋链霉菌及其制备方法 | |
CN101802168A (zh) | 非天然型抗生素的制造方法 | |
CN109810919B (zh) | 一类安莎全碳环聚酮类抗生素及其在制备抗菌药物或抗肿瘤药物中的应用 | |
CN108841769B (zh) | 一种非达霉素基因工程菌及构建方法和应用 | |
CN114875095B (zh) | 一种丙氨酰美登醇及其合成方法和应用 | |
CN114014867B (zh) | 一种安莎三烯类化合物及其制备方法与应用 | |
US20050192432A1 (en) | Derivatives of mithramycin and methods of making and uses thereof | |
CN112409372B (zh) | 玉红霉素类似物、制备方法及其应用 | |
EP0350623A2 (en) | BU-3420T antitumor antibiotic | |
CN116082362B (zh) | 一种可用于合成靶向抗肿瘤药物的氨甲酰美登醇及其合成方法与应用 | |
PAULULAT et al. | Biosynthesis of polyketomycin produced by Streptomyces diastatochromogenes Tü 6028 | |
CN109836433B (zh) | 新型LL-D49194α1类似物,及其制备方法和应用 | |
CN115161208A (zh) | 酿酒酵母基因工程菌及其生产葫芦素中间体的应用 | |
EP1443113B1 (en) | Method of obtaining indolocarbazoles using biosynthetic rebeccamycin genes | |
AU618790B2 (en) | Bu-3862t antitumor antibiotic | |
CN107541481B (zh) | 一种产表阿霉素的基因工程菌及其应用 | |
CN108864221B (zh) | 阿克拉霉素类似物及其制法和用途 | |
CN113912658B (zh) | 4’-N-demethyl-vicenistatin及其制备方法和应用 | |
CA2354030A1 (en) | Micromonospora echinospora genes encoding for biosynthesis of calicheamicin and self-resistance thereto | |
CN114540376B (zh) | 一种抗肿瘤聚酮类化合物及其制备方法和用途 | |
CN116239607B (zh) | 一种美登素衍生物及其生物合成方法与应用 | |
CN108456689A (zh) | 提高安丝菌素p-3生物合成产量的方法 | |
JP4759756B2 (ja) | 抗生物質カプラザマイシンd、g、d1、g1とその製造法 |
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 |