CN113480622A - 一种重组肺炎球菌溶血素制备及纯化的方法 - Google Patents
一种重组肺炎球菌溶血素制备及纯化的方法 Download PDFInfo
- Publication number
- CN113480622A CN113480622A CN202110896837.9A CN202110896837A CN113480622A CN 113480622 A CN113480622 A CN 113480622A CN 202110896837 A CN202110896837 A CN 202110896837A CN 113480622 A CN113480622 A CN 113480622A
- Authority
- CN
- China
- Prior art keywords
- tris
- hcl
- nacl
- edta
- purification
- 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.)
- Pending
Links
- 101710183389 Pneumolysin Proteins 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 29
- 238000000746 purification Methods 0.000 claims abstract description 59
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 48
- 239000004475 Arginine Substances 0.000 claims abstract description 27
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims abstract description 27
- 241001052560 Thallis Species 0.000 claims abstract description 17
- 230000014509 gene expression Effects 0.000 claims abstract description 17
- 239000012043 crude product Substances 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 8
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 7
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 7
- 238000002305 strong-anion-exchange chromatography Methods 0.000 claims abstract description 5
- 238000004271 weak anion exchange chromatography Methods 0.000 claims abstract description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 75
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical group Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 49
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 44
- 238000010828 elution Methods 0.000 claims description 38
- 239000011780 sodium chloride Substances 0.000 claims description 34
- 239000007853 buffer solution Substances 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 29
- 238000011282 treatment Methods 0.000 claims description 22
- 238000005406 washing Methods 0.000 claims description 22
- 238000011068 loading method Methods 0.000 claims description 21
- 238000000108 ultra-filtration Methods 0.000 claims description 21
- 238000011067 equilibration Methods 0.000 claims description 14
- 239000013604 expression vector Substances 0.000 claims description 14
- 238000004140 cleaning Methods 0.000 claims description 13
- 238000001914 filtration Methods 0.000 claims description 13
- 230000006698 induction Effects 0.000 claims description 13
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 13
- 229920002271 DEAE-Sepharose Polymers 0.000 claims description 12
- 239000000872 buffer Substances 0.000 claims description 12
- 210000004027 cell Anatomy 0.000 claims description 12
- 239000003480 eluent Substances 0.000 claims description 12
- 239000012528 membrane Substances 0.000 claims description 12
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 11
- 238000003756 stirring Methods 0.000 claims description 11
- 239000003085 diluting agent Substances 0.000 claims description 10
- 238000006073 displacement reaction Methods 0.000 claims description 10
- 238000000855 fermentation Methods 0.000 claims description 10
- 230000004151 fermentation Effects 0.000 claims description 10
- 239000012564 Q sepharose fast flow resin Substances 0.000 claims description 9
- 229930027917 kanamycin Natural products 0.000 claims description 9
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 9
- 229960000318 kanamycin Drugs 0.000 claims description 9
- 229930182823 kanamycin A Natural products 0.000 claims description 9
- 239000011148 porous material Substances 0.000 claims description 9
- 238000007865 diluting Methods 0.000 claims description 8
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 239000012466 permeate Substances 0.000 claims description 5
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 238000005352 clarification Methods 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- 239000000411 inducer Substances 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 4
- 230000009465 prokaryotic expression Effects 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 239000000047 product Substances 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 4
- 239000003054 catalyst Substances 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 abstract description 42
- 238000004458 analytical method Methods 0.000 abstract description 10
- 238000011084 recovery Methods 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 abstract 1
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- 238000001962 electrophoresis Methods 0.000 description 40
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 34
- 230000002829 reductive effect Effects 0.000 description 33
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 13
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 13
- 235000011130 ammonium sulphate Nutrition 0.000 description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 9
- 238000013368 capillary electrophoresis sodium dodecyl sulfate analysis Methods 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 239000012160 loading buffer Substances 0.000 description 8
- 102200067143 rs80357438 Human genes 0.000 description 8
- 230000027455 binding Effects 0.000 description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 230000002238 attenuated effect Effects 0.000 description 5
- 230000024203 complement activation Effects 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 241000880493 Leptailurus serval Species 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 108010073969 valyllysine Proteins 0.000 description 3
- UJGRZQYSNYTCAX-SRVKXCTJSA-N Asp-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(O)=O UJGRZQYSNYTCAX-SRVKXCTJSA-N 0.000 description 2
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 2
- JVSBYEDSSRZQGV-GUBZILKMSA-N Glu-Asp-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CCC(O)=O JVSBYEDSSRZQGV-GUBZILKMSA-N 0.000 description 2
- 108010006464 Hemolysin Proteins Proteins 0.000 description 2
- YVMQJGWLHRWMDF-MNXVOIDGSA-N Lys-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCCCN)N YVMQJGWLHRWMDF-MNXVOIDGSA-N 0.000 description 2
- 102000016387 Pancreatic elastase Human genes 0.000 description 2
- 108010067372 Pancreatic elastase Proteins 0.000 description 2
- 108010038633 aspartylglutamate Proteins 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 108010057083 glutamyl-aspartyl-leucine Proteins 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 239000003228 hemolysin Substances 0.000 description 2
- 230000002949 hemolytic effect Effects 0.000 description 2
- 108010064235 lysylglycine Proteins 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 210000001167 myeloblast Anatomy 0.000 description 2
- 229940023143 protein vaccine Drugs 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 230000003827 upregulation Effects 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- JWUZOJXDJDEQEM-ZLIFDBKOSA-N Ala-Lys-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)C)C(O)=O)=CNC2=C1 JWUZOJXDJDEQEM-ZLIFDBKOSA-N 0.000 description 1
- FEGOCLZUJUFCHP-CIUDSAMLSA-N Ala-Pro-Gln Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O FEGOCLZUJUFCHP-CIUDSAMLSA-N 0.000 description 1
- PGNNQOJOEGFAOR-KWQFWETISA-N Ala-Tyr-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=C(O)C=C1 PGNNQOJOEGFAOR-KWQFWETISA-N 0.000 description 1
- NONSEUUPKITYQT-BQBZGAKWSA-N Arg-Asn-Gly Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)NCC(=O)O)N)CN=C(N)N NONSEUUPKITYQT-BQBZGAKWSA-N 0.000 description 1
- PQWTZSNVWSOFFK-FXQIFTODSA-N Arg-Asp-Asn Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)CN=C(N)N PQWTZSNVWSOFFK-FXQIFTODSA-N 0.000 description 1
- LMPKCSXZJSXBBL-NHCYSSNCSA-N Arg-Gln-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O LMPKCSXZJSXBBL-NHCYSSNCSA-N 0.000 description 1
- HPKSHFSEXICTLI-CIUDSAMLSA-N Arg-Glu-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O HPKSHFSEXICTLI-CIUDSAMLSA-N 0.000 description 1
- FSNVAJOPUDVQAR-AVGNSLFASA-N Arg-Lys-Arg Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FSNVAJOPUDVQAR-AVGNSLFASA-N 0.000 description 1
- JZRLLSOWDYUKOK-SRVKXCTJSA-N Asn-Asp-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)N)N JZRLLSOWDYUKOK-SRVKXCTJSA-N 0.000 description 1
- DJIMLSXHXKWADV-CIUDSAMLSA-N Asn-Leu-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(N)=O DJIMLSXHXKWADV-CIUDSAMLSA-N 0.000 description 1
- GKKUBLFXKRDMFC-BQBZGAKWSA-N Asn-Pro-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O GKKUBLFXKRDMFC-BQBZGAKWSA-N 0.000 description 1
- XLDMSQYOYXINSZ-QXEWZRGKSA-N Asn-Val-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N XLDMSQYOYXINSZ-QXEWZRGKSA-N 0.000 description 1
- RGKKALNPOYURGE-ZKWXMUAHSA-N Asp-Ala-Val Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)O RGKKALNPOYURGE-ZKWXMUAHSA-N 0.000 description 1
- YDJVIBMKAMQPPP-LAEOZQHASA-N Asp-Glu-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O YDJVIBMKAMQPPP-LAEOZQHASA-N 0.000 description 1
- QHHVSXGWLYEAGX-GUBZILKMSA-N Asp-His-Gln Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)O)N QHHVSXGWLYEAGX-GUBZILKMSA-N 0.000 description 1
- TZOZNVLBTAFJRW-UGYAYLCHSA-N Asp-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)O)N TZOZNVLBTAFJRW-UGYAYLCHSA-N 0.000 description 1
- DJCAHYVLMSRBFR-QXEWZRGKSA-N Asp-Met-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@@H](N)CC(O)=O DJCAHYVLMSRBFR-QXEWZRGKSA-N 0.000 description 1
- SFJUYBCDQBAYAJ-YDHLFZDLSA-N Asp-Val-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 SFJUYBCDQBAYAJ-YDHLFZDLSA-N 0.000 description 1
- 108010059108 CD18 Antigens Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- PGPJSRSLQNXBDT-YUMQZZPRSA-N Gln-Arg-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O PGPJSRSLQNXBDT-YUMQZZPRSA-N 0.000 description 1
- UICOTGULOUGGLC-NUMRIWBASA-N Gln-Asp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)N)N)O UICOTGULOUGGLC-NUMRIWBASA-N 0.000 description 1
- GQZDDFRXSDGUNG-YVNDNENWSA-N Gln-Ile-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(O)=O GQZDDFRXSDGUNG-YVNDNENWSA-N 0.000 description 1
- RUFHOVYUYSNDNY-ACZMJKKPSA-N Glu-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O RUFHOVYUYSNDNY-ACZMJKKPSA-N 0.000 description 1
- VPKBCVUDBNINAH-GARJFASQSA-N Glu-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)O)N)C(=O)O VPKBCVUDBNINAH-GARJFASQSA-N 0.000 description 1
- FLLRAEJOLZPSMN-CIUDSAMLSA-N Glu-Asn-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N FLLRAEJOLZPSMN-CIUDSAMLSA-N 0.000 description 1
- HJIFPJUEOGZWRI-GUBZILKMSA-N Glu-Asp-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)N HJIFPJUEOGZWRI-GUBZILKMSA-N 0.000 description 1
- CKOFNWCLWRYUHK-XHNCKOQMSA-N Glu-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)N)C(=O)O CKOFNWCLWRYUHK-XHNCKOQMSA-N 0.000 description 1
- FBEJIDRSQCGFJI-GUBZILKMSA-N Glu-Leu-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O FBEJIDRSQCGFJI-GUBZILKMSA-N 0.000 description 1
- YKBUCXNNBYZYAY-MNXVOIDGSA-N Glu-Lys-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O YKBUCXNNBYZYAY-MNXVOIDGSA-N 0.000 description 1
- QDMVXRNLOPTPIE-WDCWCFNPSA-N Glu-Lys-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QDMVXRNLOPTPIE-WDCWCFNPSA-N 0.000 description 1
- KXTAGESXNQEZKB-DZKIICNBSA-N Glu-Phe-Val Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C(C)C)C(O)=O)CC1=CC=CC=C1 KXTAGESXNQEZKB-DZKIICNBSA-N 0.000 description 1
- SYAYROHMAIHWFB-KBIXCLLPSA-N Glu-Ser-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O SYAYROHMAIHWFB-KBIXCLLPSA-N 0.000 description 1
- QIZJOTQTCAGKPU-KWQFWETISA-N Gly-Ala-Tyr Chemical compound [NH3+]CC(=O)N[C@@H](C)C(=O)N[C@H](C([O-])=O)CC1=CC=C(O)C=C1 QIZJOTQTCAGKPU-KWQFWETISA-N 0.000 description 1
- JRDYDYXZKFNNRQ-XPUUQOCRSA-N Gly-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN JRDYDYXZKFNNRQ-XPUUQOCRSA-N 0.000 description 1
- WJZLEENECIOOSA-WDSKDSINSA-N Gly-Asn-Gln Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)O WJZLEENECIOOSA-WDSKDSINSA-N 0.000 description 1
- XCLCVBYNGXEVDU-WHFBIAKZSA-N Gly-Asn-Ser Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O XCLCVBYNGXEVDU-WHFBIAKZSA-N 0.000 description 1
- JMQFHZWESBGPFC-WDSKDSINSA-N Gly-Gln-Asp Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O JMQFHZWESBGPFC-WDSKDSINSA-N 0.000 description 1
- ZQIMMEYPEXIYBB-IUCAKERBSA-N Gly-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CN ZQIMMEYPEXIYBB-IUCAKERBSA-N 0.000 description 1
- GMTXWRIDLGTVFC-IUCAKERBSA-N Gly-Lys-Glu Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O GMTXWRIDLGTVFC-IUCAKERBSA-N 0.000 description 1
- OQQKUTVULYLCDG-ONGXEEELSA-N Gly-Lys-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCCCN)NC(=O)CN)C(O)=O OQQKUTVULYLCDG-ONGXEEELSA-N 0.000 description 1
- YOBGUCWZPXJHTN-BQBZGAKWSA-N Gly-Ser-Arg Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YOBGUCWZPXJHTN-BQBZGAKWSA-N 0.000 description 1
- FNXSYBOHALPRHV-ONGXEEELSA-N Gly-Val-Lys Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN FNXSYBOHALPRHV-ONGXEEELSA-N 0.000 description 1
- FYVHHKMHFPMBBG-GUBZILKMSA-N His-Gln-Asp Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N FYVHHKMHFPMBBG-GUBZILKMSA-N 0.000 description 1
- HVCRQRQPIIRNLY-IUCAKERBSA-N His-Gln-Gly Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)NCC(=O)O)N HVCRQRQPIIRNLY-IUCAKERBSA-N 0.000 description 1
- JSQIXEHORHLQEE-MEYUZBJRSA-N His-Phe-Thr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JSQIXEHORHLQEE-MEYUZBJRSA-N 0.000 description 1
- QCBYAHHNOHBXIH-UWVGGRQHSA-N His-Pro-Gly Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)C1=CN=CN1 QCBYAHHNOHBXIH-UWVGGRQHSA-N 0.000 description 1
- 241000165940 Houjia Species 0.000 description 1
- HOLOYAZCIHDQNS-YVNDNENWSA-N Ile-Gln-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N HOLOYAZCIHDQNS-YVNDNENWSA-N 0.000 description 1
- KLBVGHCGHUNHEA-BJDJZHNGSA-N Ile-Leu-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)O)N KLBVGHCGHUNHEA-BJDJZHNGSA-N 0.000 description 1
- ADDYYRVQQZFIMW-MNXVOIDGSA-N Ile-Lys-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ADDYYRVQQZFIMW-MNXVOIDGSA-N 0.000 description 1
- YKZAMJXNJUWFIK-JBDRJPRFSA-N Ile-Ser-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)O)N YKZAMJXNJUWFIK-JBDRJPRFSA-N 0.000 description 1
- PXKACEXYLPBMAD-JBDRJPRFSA-N Ile-Ser-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PXKACEXYLPBMAD-JBDRJPRFSA-N 0.000 description 1
- WKSHBPRUIRGWRZ-KCTSRDHCSA-N Ile-Trp-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)NCC(=O)O)N WKSHBPRUIRGWRZ-KCTSRDHCSA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- BQSLGJHIAGOZCD-CIUDSAMLSA-N Leu-Ala-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O BQSLGJHIAGOZCD-CIUDSAMLSA-N 0.000 description 1
- ZURHXHNAEJJRNU-CIUDSAMLSA-N Leu-Asp-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O ZURHXHNAEJJRNU-CIUDSAMLSA-N 0.000 description 1
- ZDSNOSQHMJBRQN-SRVKXCTJSA-N Leu-Asp-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N ZDSNOSQHMJBRQN-SRVKXCTJSA-N 0.000 description 1
- QDSKNVXKLPQNOJ-GVXVVHGQSA-N Leu-Gln-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O QDSKNVXKLPQNOJ-GVXVVHGQSA-N 0.000 description 1
- ZFNLIDNJUWNIJL-WDCWCFNPSA-N Leu-Glu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZFNLIDNJUWNIJL-WDCWCFNPSA-N 0.000 description 1
- HNDWYLYAYNBWMP-AJNGGQMLSA-N Leu-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(C)C)N HNDWYLYAYNBWMP-AJNGGQMLSA-N 0.000 description 1
- IEWBEPKLKUXQBU-VOAKCMCISA-N Leu-Leu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IEWBEPKLKUXQBU-VOAKCMCISA-N 0.000 description 1
- LZHJZLHSRGWBBE-IHRRRGAJSA-N Leu-Lys-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LZHJZLHSRGWBBE-IHRRRGAJSA-N 0.000 description 1
- BMVFXOQHDQZAQU-DCAQKATOSA-N Leu-Pro-Asp Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(=O)O)C(=O)O)N BMVFXOQHDQZAQU-DCAQKATOSA-N 0.000 description 1
- UCBPDSYUVAAHCD-UWVGGRQHSA-N Leu-Pro-Gly Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UCBPDSYUVAAHCD-UWVGGRQHSA-N 0.000 description 1
- YUTNOGOMBNYPFH-XUXIUFHCSA-N Leu-Pro-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O YUTNOGOMBNYPFH-XUXIUFHCSA-N 0.000 description 1
- KWLWZYMNUZJKMZ-IHRRRGAJSA-N Leu-Pro-Leu Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O KWLWZYMNUZJKMZ-IHRRRGAJSA-N 0.000 description 1
- ZJZNLRVCZWUONM-JXUBOQSCSA-N Leu-Thr-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O ZJZNLRVCZWUONM-JXUBOQSCSA-N 0.000 description 1
- MSFITIBEMPWCBD-ULQDDVLXSA-N Leu-Val-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 MSFITIBEMPWCBD-ULQDDVLXSA-N 0.000 description 1
- 208000004852 Lung Injury Diseases 0.000 description 1
- IRNSXVOWSXSULE-DCAQKATOSA-N Lys-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN IRNSXVOWSXSULE-DCAQKATOSA-N 0.000 description 1
- ZJWIXBZTAAJERF-IHRRRGAJSA-N Lys-Lys-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CCCN=C(N)N ZJWIXBZTAAJERF-IHRRRGAJSA-N 0.000 description 1
- WBSCNDJQPKSPII-KKUMJFAQSA-N Lys-Lys-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O WBSCNDJQPKSPII-KKUMJFAQSA-N 0.000 description 1
- MSSJJDVQTFTLIF-KBPBESRZSA-N Lys-Phe-Gly Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](Cc1ccccc1)C(=O)NCC(O)=O MSSJJDVQTFTLIF-KBPBESRZSA-N 0.000 description 1
- GIKFNMZSGYAPEJ-HJGDQZAQSA-N Lys-Thr-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O GIKFNMZSGYAPEJ-HJGDQZAQSA-N 0.000 description 1
- LMKSBGIUPVRHEH-FXQIFTODSA-N Met-Ala-Asn Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(N)=O LMKSBGIUPVRHEH-FXQIFTODSA-N 0.000 description 1
- HKRYNJSKVLZIFP-IHRRRGAJSA-N Met-Asn-Tyr Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O HKRYNJSKVLZIFP-IHRRRGAJSA-N 0.000 description 1
- QMIXOTQHYHOUJP-KKUMJFAQSA-N Met-Gln-Tyr Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N QMIXOTQHYHOUJP-KKUMJFAQSA-N 0.000 description 1
- NSMXRFMGZYTFEX-KJEVXHAQSA-N Met-Thr-Tyr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CCSC)N)O NSMXRFMGZYTFEX-KJEVXHAQSA-N 0.000 description 1
- WUGMRIBZSVSJNP-UHFFFAOYSA-N N-L-alanyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C)C(O)=O)=CNC2=C1 WUGMRIBZSVSJNP-UHFFFAOYSA-N 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- CDNPIRSCAFMMBE-SRVKXCTJSA-N Phe-Asn-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O CDNPIRSCAFMMBE-SRVKXCTJSA-N 0.000 description 1
- UMKYAYXCMYYNHI-AVGNSLFASA-N Phe-Gln-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N UMKYAYXCMYYNHI-AVGNSLFASA-N 0.000 description 1
- MGBRZXXGQBAULP-DRZSPHRISA-N Phe-Glu-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 MGBRZXXGQBAULP-DRZSPHRISA-N 0.000 description 1
- VXCHGLYSIOOZIS-GUBZILKMSA-N Pro-Ala-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 VXCHGLYSIOOZIS-GUBZILKMSA-N 0.000 description 1
- MRYUJHGPZQNOAD-IHRRRGAJSA-N Pro-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@@H]1CCCN1 MRYUJHGPZQNOAD-IHRRRGAJSA-N 0.000 description 1
- OFGUOWQVEGTVNU-DCAQKATOSA-N Pro-Lys-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O OFGUOWQVEGTVNU-DCAQKATOSA-N 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- RDFQNDHEHVSONI-ZLUOBGJFSA-N Ser-Asn-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O RDFQNDHEHVSONI-ZLUOBGJFSA-N 0.000 description 1
- QPFJSHSJFIYDJZ-GHCJXIJMSA-N Ser-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CO QPFJSHSJFIYDJZ-GHCJXIJMSA-N 0.000 description 1
- OLIJLNWFEQEFDM-SRVKXCTJSA-N Ser-Asp-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 OLIJLNWFEQEFDM-SRVKXCTJSA-N 0.000 description 1
- MMAPOBOTRUVNKJ-ZLUOBGJFSA-N Ser-Asp-Ser Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CO)N)C(=O)O MMAPOBOTRUVNKJ-ZLUOBGJFSA-N 0.000 description 1
- BKZYBLLIBOBOOW-GHCJXIJMSA-N Ser-Ile-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O BKZYBLLIBOBOOW-GHCJXIJMSA-N 0.000 description 1
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 1
- NIOYDASGXWLHEZ-CIUDSAMLSA-N Ser-Met-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(O)=O NIOYDASGXWLHEZ-CIUDSAMLSA-N 0.000 description 1
- WUXCHQZLUHBSDJ-LKXGYXEUSA-N Ser-Thr-Asp Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CC(O)=O)C(O)=O WUXCHQZLUHBSDJ-LKXGYXEUSA-N 0.000 description 1
- OQSQCUWQOIHECT-YJRXYDGGSA-N Ser-Tyr-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OQSQCUWQOIHECT-YJRXYDGGSA-N 0.000 description 1
- IAOHCSQDQDWRQU-GUBZILKMSA-N Ser-Val-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O IAOHCSQDQDWRQU-GUBZILKMSA-N 0.000 description 1
- SIEBDTCABMZCLF-XGEHTFHBSA-N Ser-Val-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SIEBDTCABMZCLF-XGEHTFHBSA-N 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- NJEMRSFGDNECGF-GCJQMDKQSA-N Thr-Ala-Asp Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(O)=O NJEMRSFGDNECGF-GCJQMDKQSA-N 0.000 description 1
- STGXWWBXWXZOER-MBLNEYKQSA-N Thr-Ala-His Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 STGXWWBXWXZOER-MBLNEYKQSA-N 0.000 description 1
- DGDCHPCRMWEOJR-FQPOAREZSA-N Thr-Ala-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 DGDCHPCRMWEOJR-FQPOAREZSA-N 0.000 description 1
- PQLXHSACXPGWPD-GSSVUCPTSA-N Thr-Asn-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PQLXHSACXPGWPD-GSSVUCPTSA-N 0.000 description 1
- VULNJDORNLBPNG-SWRJLBSHSA-N Thr-Glu-Trp Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N)O VULNJDORNLBPNG-SWRJLBSHSA-N 0.000 description 1
- BNGDYRRHRGOPHX-IFFSRLJSSA-N Thr-Glu-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)[C@@H](C)O)C(O)=O BNGDYRRHRGOPHX-IFFSRLJSSA-N 0.000 description 1
- QQWNRERCGGZOKG-WEDXCCLWSA-N Thr-Gly-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O QQWNRERCGGZOKG-WEDXCCLWSA-N 0.000 description 1
- FQPDRTDDEZXCEC-SVSWQMSJSA-N Thr-Ile-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O FQPDRTDDEZXCEC-SVSWQMSJSA-N 0.000 description 1
- DXPURPNJDFCKKO-RHYQMDGZSA-N Thr-Lys-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)[C@@H](C)O)C(O)=O DXPURPNJDFCKKO-RHYQMDGZSA-N 0.000 description 1
- NQQMWWVVGIXUOX-SVSWQMSJSA-N Thr-Ser-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O NQQMWWVVGIXUOX-SVSWQMSJSA-N 0.000 description 1
- IQPWNQRRAJHOKV-KATARQTJSA-N Thr-Ser-Lys Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN IQPWNQRRAJHOKV-KATARQTJSA-N 0.000 description 1
- WKGAAMOJPMBBMC-IXOXFDKPSA-N Thr-Ser-Phe Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O WKGAAMOJPMBBMC-IXOXFDKPSA-N 0.000 description 1
- BBPCSGKKPJUYRB-UVOCVTCTSA-N Thr-Thr-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O BBPCSGKKPJUYRB-UVOCVTCTSA-N 0.000 description 1
- KHTIUAKJRUIEMA-HOUAVDHOSA-N Thr-Trp-Asp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CC(O)=O)C(O)=O)=CNC2=C1 KHTIUAKJRUIEMA-HOUAVDHOSA-N 0.000 description 1
- 206010069363 Traumatic lung injury Diseases 0.000 description 1
- PNHABSVRPFBUJY-UMPQAUOISA-N Trp-Arg-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N)O PNHABSVRPFBUJY-UMPQAUOISA-N 0.000 description 1
- GKUROEIXVURAAO-BPUTZDHNSA-N Trp-Asp-Arg Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GKUROEIXVURAAO-BPUTZDHNSA-N 0.000 description 1
- DPMVSFFKGNKJLQ-VJBMBRPKSA-N Trp-Glu-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC3=CNC4=CC=CC=C43)C(=O)O)N DPMVSFFKGNKJLQ-VJBMBRPKSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- JWGXUKHIKXZWNG-RYUDHWBXSA-N Tyr-Gly-Gln Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)N[C@@H](CCC(=O)N)C(=O)O)N)O JWGXUKHIKXZWNG-RYUDHWBXSA-N 0.000 description 1
- PRONOHBTMLNXCZ-BZSNNMDCSA-N Tyr-Leu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 PRONOHBTMLNXCZ-BZSNNMDCSA-N 0.000 description 1
- ARMNWLJYHCOSHE-KKUMJFAQSA-N Tyr-Pro-Gln Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O ARMNWLJYHCOSHE-KKUMJFAQSA-N 0.000 description 1
- KRXFXDCNKLANCP-CXTHYWKRSA-N Tyr-Tyr-Ile Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 KRXFXDCNKLANCP-CXTHYWKRSA-N 0.000 description 1
- KSGKJSFPWSMJHK-JNPHEJMOSA-N Tyr-Tyr-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KSGKJSFPWSMJHK-JNPHEJMOSA-N 0.000 description 1
- PQPWEALFTLKSEB-DZKIICNBSA-N Tyr-Val-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O PQPWEALFTLKSEB-DZKIICNBSA-N 0.000 description 1
- IZFVRRYRMQFVGX-NRPADANISA-N Val-Ala-Gln Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N IZFVRRYRMQFVGX-NRPADANISA-N 0.000 description 1
- SLLKXDSRVAOREO-KZVJFYERSA-N Val-Ala-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)N)O SLLKXDSRVAOREO-KZVJFYERSA-N 0.000 description 1
- COYSIHFOCOMGCF-UHFFFAOYSA-N Val-Arg-Gly Natural products CC(C)C(N)C(=O)NC(C(=O)NCC(O)=O)CCCN=C(N)N COYSIHFOCOMGCF-UHFFFAOYSA-N 0.000 description 1
- BYOHPUZJVXWHAE-BYULHYEWSA-N Val-Asn-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N BYOHPUZJVXWHAE-BYULHYEWSA-N 0.000 description 1
- QGFPYRPIUXBYGR-YDHLFZDLSA-N Val-Asn-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N QGFPYRPIUXBYGR-YDHLFZDLSA-N 0.000 description 1
- CVIXTAITYJQMPE-LAEOZQHASA-N Val-Glu-Asn Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O CVIXTAITYJQMPE-LAEOZQHASA-N 0.000 description 1
- HQYVQDRYODWONX-DCAQKATOSA-N Val-His-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CO)C(=O)O)N HQYVQDRYODWONX-DCAQKATOSA-N 0.000 description 1
- VXDSPJJQUQDCKH-UKJIMTQDSA-N Val-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N VXDSPJJQUQDCKH-UKJIMTQDSA-N 0.000 description 1
- SYSWVVCYSXBVJG-RHYQMDGZSA-N Val-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)N)O SYSWVVCYSXBVJG-RHYQMDGZSA-N 0.000 description 1
- QRVPEKJBBRYISE-XUXIUFHCSA-N Val-Lys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)N QRVPEKJBBRYISE-XUXIUFHCSA-N 0.000 description 1
- NZYNRRGJJVSSTJ-GUBZILKMSA-N Val-Ser-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O NZYNRRGJJVSSTJ-GUBZILKMSA-N 0.000 description 1
- HTONZBWRYUKUKC-RCWTZXSCSA-N Val-Thr-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O HTONZBWRYUKUKC-RCWTZXSCSA-N 0.000 description 1
- PFMSJVIPEZMKSC-DZKIICNBSA-N Val-Tyr-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N PFMSJVIPEZMKSC-DZKIICNBSA-N 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 206010069351 acute lung injury Diseases 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 108010068265 aspartyltyrosine Proteins 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000012459 cleaning agent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 108010013768 glutamyl-aspartyl-proline Proteins 0.000 description 1
- 108010027668 glycyl-alanyl-valine Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 108010018006 histidylserine Proteins 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 1
- 108010053037 kyotorphin Proteins 0.000 description 1
- 101150109249 lacI gene Proteins 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000515 lung injury Toxicity 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 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
- 238000006384 oligomerization reaction Methods 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000007112 pro inflammatory response Effects 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 229940048914 protamine Drugs 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 108700014832 replication initiator Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 239000012465 retentate Substances 0.000 description 1
- -1 salt ion Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 108010033670 threonyl-aspartyl-tyrosine Proteins 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 108010072986 threonyl-seryl-lysine Proteins 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 108010009962 valyltyrosine Proteins 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
- C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pulmonology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种重组肺炎球菌溶血素的纯化方法,其包括以下步骤:(1)粗纯化:将精氨酸与表达所述重组肺炎球菌溶血素的菌体混合,获得粗产物;在粗纯化的体系中,所述精氨酸的终浓度为0.5~0.75M,所述菌体的终浓度为0.2~0.5g/mL;(2)精纯化:将所述粗产物经弱阴离子交换层析、疏水层析和强阴离子交换层析后,获得所述重组肺炎球菌溶血素。该方法操作便捷,过程稳定可控、目的蛋白表达量高,纯化后的目的蛋白回收率高、纯度分析仅为目标蛋白单一条带,且其各项指标均符合《中国药典》(2015版)对重组蛋白的要求,利于大规模生产。
Description
技术领域
本发明涉及一种免疫蛋白载体的制备,尤其涉及一种重组肺炎球菌溶血素表达及纯化的方法,属于生物医药及生物技术领域。
背景技术
肺炎链球菌溶血素(pneumolysin,Ply)蛋白由471个氨基酸组成的分子量约为53kDa的单链蛋白,具有较强的毒性,可通过其细胞裂解活性对人体产生损害(Mitchell等,2014;Marriott等,2008)。此外,天然形式的Ply(native Ply,nPly)蛋白还可以激活补体信号通路,引起较严重的炎症反应,造成急性肺部损伤等伤害(Calbo等,2011;Witzenrath等,2006);对于肺部损伤,目前认为也有可能是nPly直接作用于呼吸道表皮细胞的结果;还有研究显示,nPly通过呼吸道表面补体裂解、从而抑制补体的激活而达到肺炎链球菌实现定植。最近也有研究提出nPly作用于中粒性细胞,伴随中粒性细胞中弹性蛋白酶(elastase)的释放,逐渐破坏肺部免疫防御系统(Domon H等人,2016)。
nPly通过单体寡聚化成环状,在细胞膜上形成由30-50个单体聚集的孔,导致细胞通透性改变,保持内外物质发生流动,nPly能够引起对中性粒细胞和单核巨噬细胞的多种不同的促炎症反应,包括促炎症反应细胞因子的释放、激活依赖型上调β2整合素释放以及增加的钙离子上调。溶血活性与补体激活效应区位于结构域4的两个不同区域上。结构域4羧基末端存在一个色氨酸丰富环(Trp-rich loop),nPly的胆固醇结合及膜插入部位被确认在该结构域4的羧基末端——该区域在巯基激活的溶血素中属于保守序列。针对该部位的氨基酸删除和突变或者以该位置羟基末端为靶点的单克隆抗体结合都能够使nPly的溶血活性丧失。Trp-rich loop长达11个氨基酸,包含有3个色氨酸,并且被认为是nPly作为溶血素由成孔前状态转化到成孔状态的必需构造。此外,补体激活区域也被确定在结构域4位置上。在nPly和C端重激活蛋白的相同氨基酸位置进行突变,能同时降低补体激活以及免疫球蛋白结合活性。由于天然的nPly蛋白所具有的毒性,降低其所具有的不利作用就变得极有意义。
对于理想的疫苗应用要求来说,nPly应该同时缺少其细胞毒性及补体激活效应(侯宏嘉等人,2014)。由于结构和功能的关系越来越清晰,已有研究已经证明,采用重组突变可以去除nPly蛋白毒性,同时保留其抗原性(Kirkham等,2006)。Kamtchoua等(KamtchouaT等,2013)研究表明nPly减毒衍生物(PlyD1)具有安全性和免疫原性,可产生中和性抗体IgG,是一种重要的候选蛋白疫苗。
基于nPly的作用,关于nPly减毒衍生物的制备方法也成为蛋白疫苗研究的热点,根据已有报道,关于nPly减毒衍生物的制备方法复杂且方法传统单一,因此需要结合目前生物技术发展前景较好的方法开发出操作简单、可放大、可重复、过程可控、稳定的重组肺炎球菌溶血素表达及纯化方法,以获得高质量的载体蛋白。
本领域一般在保存蛋白时,需添加吐温80表面活性剂等方式。
发明内容
为了解决关于nPly减毒衍生物的制备方法复杂且方法传统单一等上述问题,本发明提供了一种重组肺炎球菌溶血素高效表达、有效提取和高质量分离的表达及纯化方法。
为解决上述技术问题,本发明提供的技术方案之一为:一种重组肺炎球菌溶血素的纯化方法,其包括以下步骤:
(1)粗纯化:将精氨酸与表达所述重组肺炎球菌溶血素的菌体混合,获得粗产物;在粗纯化的体系中,所述精氨酸的终浓度为0.5~0.75M,所述菌体的终浓度为0.2~0.5g/mL,例如0.3g/mL;所述重组肺炎球菌溶血素的氨基酸序列如SEQ ID NO:2所示;
(2)精纯化:将所述粗产物经弱阴离子交换层析、疏水层析和强阴离子交换层析后,获得所述重组肺炎球菌溶血素;
所述弱阴离子交换层析的介质使用DEAE Sepharose FF,所述疏水层析的介质使用Phenyl Bestarose HP,所述强阴离子交换层析的介质使用Q Sepharose FF。
在某一具体实施例中,步骤(1)包括:
(a)高渗处理:将菌体与精氨酸进行混合,进行搅拌;所述搅拌的时长为1~5小时例如3小时;
(b)低渗处理:使用稀释剂稀释,进行搅拌,获得稀释液;所述稀释剂例如为Tris-HCl,所述搅拌时长为12~20小时;
(c)超滤澄清:将步骤(c)中所述稀释液进行过滤,收集透过液;所述过滤使用的膜包孔径例如为0.22~0.45μm。
优选地,步骤(c)后还包括(d)超滤浓缩:将透过液进行超滤浓缩,收集截流液,将截流液使用置换液置换例如4次,获得粗制的重组肺炎球菌溶血素(rPly);所述置换液为Tris-HCl缓冲液;和/或,步骤(a)前还包括含有所述菌体的发酵液进行浓缩、过滤,获得浓缩的菌体;所述过滤使用的膜包孔径为0.22~0.45μm。
更优选地,步骤(a)中浓缩的菌体的浓度为0.3g/ml,所述精氨酸终浓度为0.5mol/L;步骤(b)中所述的稀释剂为50mM Tris-HCl、pH8.0,所述的稀释的倍数为10倍;和/或,步骤(c)中所述超滤浓缩使用的膜包孔径为10Kda~30Kda,所述的置换液为20mM Tris-HCl、pH8.0、含1mM EDTA和0.062M NaCl,和/或,所述的置换液和截流液的体积比为5:1。
在某一具体实施例中,步骤(2)包括:
(2-1)将步骤(1)获得的粗产品上样于经平衡后的DEAE Sepharose FF,清洗、洗脱后收集洗脱液1;所述平衡、所述清洗和所述洗脱使用含NaCl的Tris-HCl缓冲液1,pH8.0-8.3优选8.0;所述平衡和所述清洗中使用的NaCl终浓度为0~0.1M;所述洗脱中使用的NaCl终浓度为0.1~0.2M优选0.12M;
(2-2)将上述步骤(2-1)中的洗脱液1与(NH4)2SO4混合获得混合液1,(NH4)2SO4终浓度为0.2~0.6M,将上述混合液上样于经平衡后的Phenyl Bestarose HP疏水层析柱,清洗、洗脱后收集洗脱液2;所述平衡和所述清洗使用含0.2~0.6M(NH4)2SO4的PB缓冲液;
(2-3)将上述步骤(2-2)中的洗脱液2稀释至电导率<5.0mS/cm后上样于经平衡后的Q Sepharose FF,清洗、洗脱后收集洗脱液3,所述稀释、所述平衡和所述清洗使用Tris-HCl缓冲液2,pH8.0-8.5优选pH8.5;所述洗脱使用含终浓度为0.04~0.13M NaCl的Tris-HCl缓冲液2。
优选地,步骤(2-1)中所述平衡和所述清洗使用20mM Tris-HCl、pH8.0、含1mMEDTA和0.062M NaCl,所述洗脱使用20mM Tris-HCl、pH8.0、含1mM EDTA和0.12M NaCl;
步骤(2-2)中所述平衡和所述清洗使用20mM PB、pH7.5、含1mM EDTA、0.6M(NH4)2SO4,所述洗脱使用20mM PB、pH7.5、含1mM EDTA;
步骤(2-3)中所述平衡、所述稀释和所述清洗使用100mM Tris-HCl、pH8.5、含1mMEDTA,所述洗脱使用100mM Tris-HCl、pH8.5、含1mM EDTA和0.12M NaCl。
在某一具体实施例中,还包括:
(2-4)将上述步骤(2-3)中的洗脱液3进行超滤浓缩、置换,获得精制rPly;所述超滤浓缩使用的超滤膜包孔径为10Kda~30Kda;和/或,所述置换使用硼酸盐缓冲液。
优选地,所述置换使用含0.9%NaCl的200mM硼酸盐缓冲液、pH8.0。
在某一具体实施例中,还包括:
(2-5)将上述步骤(2-4)中获得的精制rPly过滤后,分装保存至温度2℃~﹣80℃。
优选地,所述过滤使用0.22μm滤器,和/或,所述分装保存温度为﹣80℃。
在某一具体实施例中,步骤(2-1)中所述平衡和所述清洗分别使用2倍柱体积和3倍柱体积的20mM Tris-HCl、pH8.0、含1mM EDTA和0.062M NaCl、所述洗脱使用3倍柱体积的20mM Tris-HCl、pH8.0、含1mM EDTA和0.12M NaCl;
步骤(2-2)中所述平衡和所述清洗分别使用2倍柱体积和3倍柱体积的20mM PB、pH7.5、含1mM EDTA、0.6M(NH4)2SO4、所述洗脱使用3倍柱体积的20mM PB、pH7.5、含1mMEDTA;
步骤(2-3)中所述平衡和所述清洗分别使用2倍柱体积和3倍柱体积的100mMTris-HCl、pH8.5、含1mM EDTA、所述洗脱使用3倍柱体积的100mM Tris-HCl、pH8.5、含1mMEDTA和0.12M NaCl;
步骤(2-4)中所述置换使用10倍于洗脱液3的体积的200mM硼酸盐、pH8.0、含0.9%NaCl。
在某一具体实施例中,其包括以下步骤:
(I)培养表达氨基酸序列如SEQ ID NO:2所示的重组肺炎球菌溶血素的宿主细胞,所述宿主细胞为真核细胞或原核细胞;
(II)当OD600nm值例如为≥1,加入诱导剂进行诱导表达后即得。
优选地,所述重组肺炎球菌溶血素由核苷酸序列如SEQ ID NO:1所示的基因编码。
更优选地,步骤(I)中所述培养使用含有卡那霉素的三角瓶或发酵罐,步骤(II)中所述的诱导剂为IPTG;和/或,所述培养的温度为34~37℃,所述培养的pH为6.9~7.1,所述IPTG的浓度为0.1~1mM,所述诱导的温度为20~37℃,所述诱导的时间为2~6小时或过夜。
进一步更优选地,所述培养的温度为35℃,所述培养的pH为7.0,所述卡那霉素的终浓度为30μg/ml,所述IPTG的浓度为0.1mM或0.3mM,所述诱导的温度为32℃,所述诱导的时间为4小时。
在某一具体实施例中,所述原核细胞为大肠杆菌。
优选地,所述大肠杆菌为BL21(DE3)菌株。
在某一具体实施例中,所述宿主细胞包括表达载体,所述表达载体包含编码氨基酸序列如SEQ ID NO:2所示的重组肺炎球菌溶血素的核苷酸。
优选地,所述表达载体为原核表达载体或真核表达载体。
更优选地,所述原核表达载体为pET30a。
为解决上述技术问题,本发明提供的技术方案之二为:一种氨基酸序列如SEQ IDNO:2所示的重组肺炎球菌溶血素的保存方法,在-80±10℃条件下,使用含0.9%NaCl溶液的pH 8.0、0.2mol/L硼酸盐缓冲液保存所述重组肺炎球菌溶血素。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:
本发明提供了核苷酸序列得到的nPly基因减毒突变体(Reconbinant Ply,rPly)编码序列,合成后的基因序列经双酶切后连接入表达载体pET30a(+)中,rPly在大肠杆菌BL21(DE3)中获得了可溶性高表达,目的蛋白占碎菌上清总蛋白的约30%以上。经DEAESepharose FF柱、Phenyl Bestarose HP柱和Q Sepharose FF柱三步纯化后,目的蛋白纯度可达95%以上,精蛋白产率在200mg/L发酵液以上,细菌内毒素<0.1EU/μg。
本发明在-80±10℃条件下,缓冲液为pH 8.0的0.2mol/L硼酸盐缓冲液含0.9%NaCl溶液时,rPly蛋白外观均澄清且SDS-PAGE电泳显示无降解和聚合现象出现,此条件可用于rPly蛋白的长期保存。
附图说明
图1为表达载体pET30a-Ply构建示意图。
图2为经粗纯化后和三步纯化过程中rPly还原型SDS-PAGE电泳图谱;其中,泳道1为粗纯化后的rPly、泳道2为DEAE Sepharose FF纯化后的rPly、泳道3为Phenyl BestaroseHP纯化后的rPly、泳道4为Q Sepharose FF纯化后的rPly、泳道5为超滤置换及浓缩后的rPly。
图3为粗纯化使用不同试剂结果的比较;其中泳道1为0.5M精氨酸+50mM Tris-HCl(pH8.0)处理,泳道2为0.5M甘氨酸+50mM Tris-HCl(pH8.0)处理,泳道3为20%蔗糖+50mMTris-HCl(pH8.0)处理。
图4为经精纯化后得到的rPly蛋白还原型和非还原型SDS-PAGE电泳图谱与分析;其中,图4的A为rPly蛋白还原型和非还原型SDS-PAGE电泳图谱,泳道1、2、3为非还原型SDS-PAGE电泳,泳道4、5、6为还原型SDS-PAGE电泳;图4的B为rPly非还原型SDS-PAGE电泳纯度分析;图4的C为rPly非还原型SDS-PAGE电泳纯度分析。
图5为经精纯化后得到的rPly蛋白还原型和非还原型CE-SDS电泳图谱;其中,图5的A为非还原型CE-SDS电泳整体图谱,图5的B为非还原型CE-SDS电泳局部放大图谱,图5的C为非还原型CE-SDS电泳纯度分析;图5的D为还原型CE-SDS电泳整体图谱,图5的E为还原型CE-SDS电泳局部放大图谱,图5的F为还原型CE-SDS电泳纯度分析。
图6为粗制样品不同处理方式的rPly SDS-PAGE电泳图,均为还原SDS-PAGE电泳;其中,lane1、3为0.5M精氨酸高渗破碎菌体,lane2、4为0.75M精氨酸高渗破碎菌体,lane5、7为0.5M精氨酸高渗破碎上清,lane6、8为0.75M精氨酸高渗破碎上清,lane9为0.5M精氨酸高、低渗处理后收集的粗制rPly的还原型SDS-PAGE电泳,lane10为0.5M精氨酸高、低渗处理后收集的粗制rPly的非还原型SDS-PAGE电泳。
图7为DEAE Sepharose FF纯化时不同pH缓冲液条件下层析图与对应的电泳图;图7的A为pH 8.0缓冲液条件下层析图(方框标识位置为Ply洗脱峰),图7的B为pH 8.0缓冲液条件下Ply洗脱峰对应SDS-PAGE电泳图;图7的C为pH 8.3缓冲液条件下层析图(方框标识位置为Ply洗脱峰),图7的D为pH 8.3缓冲液条件下Ply洗脱峰对应SDS-PAGE电泳图;图7的E为pH 8.5缓冲液条件下层析图(方框标识位置为Ply洗脱峰),图7的F为pH 8.5缓冲液条件下Ply洗脱峰对应SDS-PAGE电泳图。
图8为Phenyl Bestarose HP纯化时不同硫酸铵上样浓度纯化后的层析图与电泳图;图8的A为1mol/L(NH4)2SO4上样浓度纯化后的层析图;图8的B为0.6mol/L(NH4)2SO4上样浓度纯化后的层析图;图8的C为0.2mol/L(NH4)2SO4上样浓度纯化后的层析图;图8的D为不同浓度硫酸铵的上样缓冲液纯化后的非还原SDS-PAGE电泳图,其中1-3分别为1mol/L、0.6mol/L和0.2mol/L(NH4)2SO4上样纯化后的流穿样品;4-6分别为1mol/L、0.6mol/L和0.2mol/L(NH4)2SO4上样,用不含硫酸铵缓冲液洗脱的样品。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
实施例1重组肺炎球菌溶血素的制备
1.1.表达载体pET-30a-rPly的构建
将目的蛋白rPly(T65C,G293C,C428A)基因,连接入用NdeI和XhoI双酶切后的表达载体pET30a(+)上,经转化、培养、筛选后测序。测序正确的质粒命名为pET30a-Ply(载体构建示意图见图1)。
rPly(T65C,G293C,C428A)核苷酸序列(SEQ ID NO:1):
ATGGCAAATAAAGCAGTAAATGACTTTATACTAGCTATGAATTACGATAAAAAGAAACTCTTGACCCATCAGGGAGAAAGTATTGAAAATCGTTTCATCAAAGAGGGTAATCAGCTACCCGATGAGTTTGTTGTTATCGAAAGAAAGAAGCGGAGCTTGTCGACAAATACAAGTGATATTTCTGTAACAGCTTGCAACGACAGTCGCCTCTATCCTGGAGCACTTCTCGTAGTGGATGAGACCTTGTTAGAGAATAATCCCACTCTTCTTGCGGTTGATCGTGCTCCGATGACTTATAGTATTGATTTGCCTGGTTTGGCAAGTAGCGATAGCTTTCTCCAAGTGGAAGACCCCAGCAATTCAAGTGTTCGCGGAGCGGTAAACGATTTGTTGGCTAAGTGGCATCAAGATTATGGTCAGGTCAATAATGTCCCAGCTAGAATGCAGTATGAAAAAATAACGGCTCACAGCATGGAACAACTCAAGGTCAAGTTTGGTTCTGACTTTGAAAAGACAGGGAATTCTCTTGATATTGATTTTAACTCTGTCCATTCAGGTGAAAAGCAGATTCAGATTGTTAATTTTAAGCAGATTTATTATACAGTCAGCGTAGACGCTGTTAAAAATCCAGGAGATGTGTTTCAAGATACTGTAACGGTAGAGGATTTAAAACAGAGAGGAATTTCTGCAGAGCGTCCTTTGGTCTATATTTCGAGTGTTGCTTATGGGCGCCAAGTCTATCTCAAGTTGGAAACCACGAGTAAGAGTGATGAAGTAGAGGCTGCTTTTGAAGCTTTGATAAAAGGAGTCAAGGTAGCTCCTCAGACAGAGTGGAAGCAGATTTTGGACAATACAGAAGTGAAGGCGGTTATTTTATGCGGCGACCCAAGTTCGGGTGCCCGAGTTGTAACAGGCAAGGTGGATATGGTAGAGGACTTGATTCAAGAAGGCAGTCGCTTTACAGCAGATCATCCAGGCTTGCCGATTTCCTATACAACTTCTTTTTTACGTGACAATGTAGTTGCGACCTTTCAAAACAGTACAGACTATGTTGAGACTAAGGTTACAGCTTACAGAAACGGAGATTTACTGCTGGATCATAGTGGTGCCTATGTTGCCCAATATTATATTACTTGGGATGAATTATCCTATGATCATCAAGGTAAGGAAGTCTTGACTCCTAAGGCTTGGGACAGAAATGGGCAGGATTTGACGGCTCACTTTACCACTAGTATTCCTTTAAAAGGGAATGTTCGTAATCTCTCTGTCAAAATTAGAGAGGCGACCGGGCTTGCCTGGGAATGGTGGCGTACGGTTTATGAAAAAACCGATTTGCCACTAGTGCGTAAGCGGACGATTTCTATTTGGGGAACAACTCTCTATCCTCAGGTAGAGGATAAGGTAGAAAATGACTAG
rPly(T65C,G293C,C428A)氨基序列(SEQ ID NO:2):
MANKAVNDFI LAMNYDKKKL LTHQGESIEN RFIKEGNQLP DEFVVIERKK RSLSTNTSDI
SVTACNDSRL YPGALLVVDE TLLENNPTLL AVDRAPMTYS IDLPGLASSD SFLQVEDPSN
SSVRGAVNDL LAKWHQDYGQ VNNVPARMQY EKITAHSMEQ LKVKFGSDFE KTGNSLDIDF
NSVHSGEKQI QIVNFKQIYY TVSVDAVKNP GDVFQDTVTV EDLKQRGISA ERPLVYISSV
AYGRQVYLKL ETTSKSDEVE AAFEALIKGV KVAPQTEWKQ ILDNTEVKAV ILCGDPSSGA
RVVTGKVDMV EDLIQEGSRF TADHPGLPIS YTTSFLRDNV VATFQNSTDY VETKVTAYRN
GDLLLDHSGA YVAQYYITWD ELSYDHQGKE VLTPKAWDRN GQDLTAHFTT SIPLKGNVRN
LSVKIREATG LAWEWWRTVY EKTDLPLVRK RTISIWGTTL YPQVEDKVEN D
rPly(T65C,G293C,C428A)目的基因序列引物:
CPUEM-A-F:5’-TAAGAAGGAGATATACATATGGCGAACAAAGCTGTGAACGATTTC-3’(SEQ IDNO:3)
CPUEM-A-R:5’-CAGTGGTGGTGGTGGTGGTGCTCGAGTTAGTCGTTCTCAACTTTG-3’(SEQ IDNO:4)
pET30a(+)质粒包含质粒复制起始子,卡那霉素抗性基因Kan,lacI序列。
1.2.rPly表达工程菌构建
将1.1中鉴定正确的阳性重组质粒转化至宿主细胞BL(DE3)中表达,进行筛选、鉴定及表达,具体包括目的蛋白的诱导表达、目的蛋白聚丙烯酰胺SDS-PAGE电泳分析;将表达目的蛋白的菌株进行测序鉴定重组质粒的正确性。
1.3.rPly的工程菌表达
将rPly的工程菌涂布于含有卡那霉素的LB平板,37℃培养过夜;挑取单克隆再次涂布含有卡那霉素的LB平板,37℃培养过夜;将菌落接种至含卡那霉素LB培养基的三角瓶中,35℃培养;卡那霉素终浓度为30μg/ml;OD600nm≈1.0时进行28℃诱导表达,IPTG终浓度为0.3mM;诱导表达后的rPly为可溶性蛋白。
1.4.rPly的发酵
将重组肺炎球菌溶血素表达菌株大肠杆菌BL21(DE3)pET30a-rPly进行30L发酵罐20L发酵体积发酵时,pH为6.9~7.1,培养温度为34~36℃,待OD600nm≥10时,进行31~33℃诱导,IPTG浓度为0.1mM,诱导时长为4h。
30L发酵罐20L发酵体积发酵,rPly平均蛋白表达量1.172g/L。
1.5.rPly的粗纯化
rPly的粗纯化包括:(1)菌体浓缩:将发酵液进行菌体浓缩,菌体浓度不高于0.6g/ml,膜包孔径为0.45μm,转速为200~500rpm;(2)精氨酸高渗处理:在(1)样品中加入精氨酸粉末,精氨酸终浓度为0.5M,菌体终浓度为0.3g/ml,高渗搅拌3h;(3)低渗处理:将(2)处理样品用50mM Tris-HCl(pH8.0),含1mM EDTA进行10倍稀释,低渗搅拌>12h;(4)超滤澄清:将(3)中稀释后的样品进行超滤澄清,膜包孔径为0.45μm,收集透过液;(5)超滤浓缩:将(4)中样品进行超滤浓缩,膜包孔径为10kDa,收集截留液,用20mM Tris-HCl(pH8.0),含1mMEDTA和0.062M NaCl进行5倍体积置换4次,收获粗制rPly,纯度67.11%(SDS-PAGE电泳图见图2,其中,泳道1为粗纯化后的rPly、泳道2为DEAE Sepharose FF纯化后的rPly、泳道3为Phenyl Bestarose HP纯化后的rPly、泳道4为Q Sepharose FF纯化后的rPly)。
1.6.粗纯化使用不同试剂与处理方式结果的比较
不同试剂结果的比较:在室温条件下,分别用20%蔗糖+50mM Tris-HCl(pH8.0)、0.5M甘氨酸+50mM Tris-HCl(pH8.0)、0.5M精氨酸+50mM Tris-HCl(pH8.0)溶解菌体至2g/ml,待菌体分散均匀后,分别补加以上溶液,稀释菌体浓度为2g/ml,660rpm搅拌均匀,2~8℃放置2h,7000rpm离心收集沉淀,将沉淀用50mM Tris-HCl(pH8.0溶解至0.1g/ml,冰浴30min后,分别12000rpm离心5min,收集上清液。结果如图3显示:在处理体积和处理浓度是一致的情况下,经精氨酸处理后rPly释放量较甘氨酸、蔗糖处理高(泳道蛋白条带较粗)。
不同处理方式的比较:分别进行高渗处理与均质机破碎处理,高渗处理条件同1.5,均质机处理条件为:900Psi,破碎3次。见图6,结果显示:经精氨酸高渗破碎处理后,大部分Ply释放于上清液中,少量损失于在破碎菌体中;0.75M精氨酸高渗破碎处理与0.5M精氨酸高渗破碎释放出的Ply量无明显差异;0.5M精氨酸高低渗与0.5M精氨酸高渗破碎两种处理比较,两者均释放出大量的Ply,但后者同时也释放出较多的杂质(核酸、杂蛋白等)。由上可见,0.5M精氨酸高低渗处理粗制Ply效果最佳。
1.7 rPly的精纯化
第一步纯化:将粗纯化获得的rPly,进行DEAE Sepharose FF纯化。(1)平衡:用20mM Tris-HCl(pH8.0),含1mM EDTA和0.062M NaCl平衡2CV;(2)上样:上样粗制rPly,待上样完毕后,再用20mM Tris-HCl(pH8.0),含1mM EDTA和0.062M NaCl清洗3CV;(3)洗脱:用20mM Tris-HCl(pH8.0),含1mM EDTA和0.12M NaCl洗脱3CV,从起峰收集至峰落位置;(4)在位清洗(clean in position,CIP):用1.5M NaCl的清洁液1和0.5M NaOH的清洁液2各清洁2CV;(5)再生及平衡:用1.5M NaCl再生1CV,再用20mM Tris-HCl(pH8.0),含1mM EDTA和0.062M NaCl平衡4CV。
第二步纯化:将DEAE Sepharose FF纯化获得的rPly,进行Phenyl Bestarose HP纯化。(1)平衡:用20mM PB(pH7.5),含1mM EDTA和0.6M(NH4)2SO4平衡2CV;(2)上样:样品与2M(NH4)2SO4按照2.3∶1比例混合后上样,待上样完毕后,再用20mM PB(pH7.5),含1mM EDTA和0.6M(NH4)2SO4清洗3CV;(3)洗脱:用20mM PB(pH7.5),含1mM EDTA洗脱3CV,从起峰收集至峰落位置;(4)CIP:用H2O的清洁液3和0.5M NaOH的清洁液2各清洁2CV;(5)再生及平衡:用H2O再生1CV,再用20mM PB(pH7.5),含1mM EDTA和0.06M(NH4)2SO4平衡4CV。
第三步纯化:将Phenyl Bestarose HP纯化获得的rPly,进行Q Sepharose FF纯化。(1)平衡:用100mM Tris-HCl(pH8.5),含1mM EDTA平衡2CV;(2)上样:样品用100mMTris-HCl(pH8.5),含1mM EDTA稀释至电导率<5.0mS/cm后上样,待上样完毕后,再用100mMTris-HCl(pH8.5),含1mM EDTA清洗3CV;(3)洗脱:用100mM Tris-HCl(pH8.5),含1mM EDTA和0.12M NaCl洗脱3CV,从起峰收集至峰落位置;(4)CIP:用1.5M NaCl的清洁液1和0.5MNaOH的清洁液2各清洁2CV;(5)再生及平衡:用1.5M NaCl再生1CV,再用100mM Tris-HCl(pH8.5),含1mM EDTA平衡4CV。
超滤:将Q Sepharose FF纯化获得的rPly进行超滤浓缩及置换。超滤膜包孔径为10kDa,将样品浓缩后,用200mM硼酸盐(pH8.0),含0.9%NaCl缓冲液进行10倍体积三次置换,收集精制rPly。置换后的A280nm>13.6以上时,精制rPly蛋白浓度为10mg/ml以上。
保存:将精制rPly用0.22μm滤器过滤后,分装保存至﹣80℃±10℃。
结果显示,纯化过程中,第一步纯化后的rPly纯度在80%以上,第二步、第三步纯化后的rPly纯度在95%以上(SDS-PAGE电泳图见图2,其中,泳道1为粗纯化后的rPly、泳道2为DEAE Sepharose FF纯化后的rPly、泳道3为Phenyl Bestarose HP纯化后的rPly、泳道4为Q Sepharose FF纯化后的rPly)。精纯化后获得rPly经还原型和非还原型SDS-PAGE电泳、还原型和非还原型CE-SDS电泳分析纯度均在98%以上(SDS-PAGE电泳图见图4,其中,图4的A为rPly蛋白还原型和非还原型SDS-PAGE电泳图谱,泳道1、2、3为非还原型SDS-PAGE电泳,泳道4、5、6为还原型SDS-PAGE电泳;图4的B为rPly非还原型SDS-PAGE电泳纯度分析;图4的C为rPly非还原型SDS-PAGE电泳纯度分析;CE-SDS电泳见图5,其中,图5的A、B、C为非还原型,图5的D、E、F为还原型),细菌内毒素≤0.01EU/μg,宿主蛋白残留量<0.1%,外源性DNA残留量<0.01ng/μg。
实施例2 对比例
2.1.DEAE Sepharose FF纯化
样品预先分别用pH为8.0、8.3和8.5的缓冲液5倍稀释后上样,分别用pH为8.0、8.3和8.5的缓冲液0.02mol/L Tris-HCl+0.001mol/L EDTA清洗层析柱5CV,至A280nm为基线,再进行0-35%1M NaCl线性梯度洗脱,分布收集样品通过SDS-PAGE电泳比较pH8.0、8.3和8.5缓冲液条件下的纯化效果。
不同pH缓冲液条件下的Ply起峰电导及杂质起峰电导
层析图谱及SDS-PAGE电泳图如图7所示,图7的A为pH 8.0缓冲液条件下层析图(方框标识位置为Ply洗脱峰),图7的B为pH 8.0缓冲液条件下Ply洗脱峰对应SDS-PAGE电泳图;图7的C为pH 8.3缓冲液条件下层析图(方框标识位置为Ply洗脱峰),图7的D为pH 8.3缓冲液条件下Ply洗脱峰对应SDS-PAGE电泳图;图7的E为pH 8.5缓冲液条件下层析图(方框标识位置为Ply洗脱峰),图7的F为pH 8.5缓冲液条件下Ply洗脱峰对应SDS-PAGE电泳图。
缓冲液pH分别为8.0和8.3时,杂蛋白和目标蛋白起峰位置(电导)基本无差异,但前者目标峰与杂蛋白峰完全分离,rPly回收率约为75.91%,后者目标峰起峰与杂质峰峰顶重叠,峰分离效果降低,rPly回收率约为70.85%;缓冲液pH 8.5时,杂质峰和目标蛋白洗脱峰出峰位置(洗脱电导)均后移,且两峰峰顶重合,rPly回收率仅约为42.67%,可见纯化效果(分辨率)极其不理想。综上,缓冲液pH为8.0±0.05时,目标蛋白与杂蛋白分离效果最佳,rPly蛋白回收率较高。
2.2 Phenyl Bestarose HP纯化
分别用含不同浓度(NH4)2SO4的缓冲液作为上样缓冲液,即硫酸铵浓度分别为1mol/L、0.6mol/L、0.2mol/L,将样品分别与2mol/L、1.2mol/L、0.4mol/L硫酸铵溶液按1∶1体积比例混合,在柱直径为1cm,柱高为20cm的层析柱上,上样约3CV后,分别用含1mol/L、0.6mol/L、0.2mol/L(NH4)2SO4的0.02mol/L PB+0.001mol/L EDTA(pH 7.5)缓冲液清洗3CV,再分别进行步阶洗脱3CV。
层析图谱及SDS-PAGE电泳图如图8所示,图8的A为1mol/L(NH4)2SO4上样浓度纯化后的层析图,图8的B为0.6mol/L(NH4)2SO4上样浓度纯化后的层析图,图8的C为0.2mol/L(NH4)2SO4上样浓度纯化后的层析图,图8的D为不同浓度硫酸铵的上样缓冲液纯化SDS-PAGE电泳图。由层析图谱结合SDS-PAGE电泳结果,上样缓冲液中(NH4)2SO4浓度分别为1mol/L、0.6mol/L、0.2mol/L时,流穿液中的杂蛋白条带逐渐增多,但无目标蛋白,可见当上样缓冲液中(NH4)2SO4浓度在0.2mol/L以上时,rPly处于吸附状态,且用不含有硫酸铵的缓冲液洗脱后,rPly纯度基本一致。将样品浓度提高,硫酸铵浓度为1mol/L时,样品中产生大量的白色絮状物沉淀,可能由于硫酸铵浓度过高导致其他杂蛋白变性,为防止上样过程中层析柱堵塞,故样品硫酸铵终浓度应在1mol/L以下;当上样缓冲液硫酸铵浓度为0.6mol/L时,rPly最大结合量为43mg/ml(gel);当上样缓冲液硫酸铵浓度为0.2mol/L时,rPly最大结合量为11mg/ml(gel)。根据以上研究可知,上样缓冲液中硫酸铵浓度的提高有利于rPly蛋白在Phenyl Bestarose HP介质上结合量的提高,且当硫酸铵浓度为0.6mol/L时rPly结合量最大,即为最佳的上样缓冲液盐离子浓度。
SEQUENCE LISTING
<110> 江苏坤力生物制药有限责任公司
<120> 一种重组肺炎球菌溶血素制备及纯化的方法
<130> P21014219C
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1416
<212> DNA
<213> Artificial Sequence
<220>
<223> rPly(T65C,G293C,C428A)核苷酸序列
<400> 1
atggcaaata aagcagtaaa tgactttata ctagctatga attacgataa aaagaaactc 60
ttgacccatc agggagaaag tattgaaaat cgtttcatca aagagggtaa tcagctaccc 120
gatgagtttg ttgttatcga aagaaagaag cggagcttgt cgacaaatac aagtgatatt 180
tctgtaacag cttgcaacga cagtcgcctc tatcctggag cacttctcgt agtggatgag 240
accttgttag agaataatcc cactcttctt gcggttgatc gtgctccgat gacttatagt 300
attgatttgc ctggtttggc aagtagcgat agctttctcc aagtggaaga ccccagcaat 360
tcaagtgttc gcggagcggt aaacgatttg ttggctaagt ggcatcaaga ttatggtcag 420
gtcaataatg tcccagctag aatgcagtat gaaaaaataa cggctcacag catggaacaa 480
ctcaaggtca agtttggttc tgactttgaa aagacaggga attctcttga tattgatttt 540
aactctgtcc attcaggtga aaagcagatt cagattgtta attttaagca gatttattat 600
acagtcagcg tagacgctgt taaaaatcca ggagatgtgt ttcaagatac tgtaacggta 660
gaggatttaa aacagagagg aatttctgca gagcgtcctt tggtctatat ttcgagtgtt 720
gcttatgggc gccaagtcta tctcaagttg gaaaccacga gtaagagtga tgaagtagag 780
gctgcttttg aagctttgat aaaaggagtc aaggtagctc ctcagacaga gtggaagcag 840
attttggaca atacagaagt gaaggcggtt attttatgcg gcgacccaag ttcgggtgcc 900
cgagttgtaa caggcaaggt ggatatggta gaggacttga ttcaagaagg cagtcgcttt 960
acagcagatc atccaggctt gccgatttcc tatacaactt cttttttacg tgacaatgta 1020
gttgcgacct ttcaaaacag tacagactat gttgagacta aggttacagc ttacagaaac 1080
ggagatttac tgctggatca tagtggtgcc tatgttgccc aatattatat tacttgggat 1140
gaattatcct atgatcatca aggtaaggaa gtcttgactc ctaaggcttg ggacagaaat 1200
gggcaggatt tgacggctca ctttaccact agtattcctt taaaagggaa tgttcgtaat 1260
ctctctgtca aaattagaga ggcgaccggg cttgcctggg aatggtggcg tacggtttat 1320
gaaaaaaccg atttgccact agtgcgtaag cggacgattt ctatttgggg aacaactctc 1380
tatcctcagg tagaggataa ggtagaaaat gactag 1416
<210> 2
<211> 471
<212> PRT
<213> Artificial Sequence
<220>
<223> rPly(T65C,G293C,C428A)氨基酸序列
<400> 2
Met Ala Asn Lys Ala Val Asn Asp Phe Ile Leu Ala Met Asn Tyr Asp
1 5 10 15
Lys Lys Lys Leu Leu Thr His Gln Gly Glu Ser Ile Glu Asn Arg Phe
20 25 30
Ile Lys Glu Gly Asn Gln Leu Pro Asp Glu Phe Val Val Ile Glu Arg
35 40 45
Lys Lys Arg Ser Leu Ser Thr Asn Thr Ser Asp Ile Ser Val Thr Ala
50 55 60
Cys Asn Asp Ser Arg Leu Tyr Pro Gly Ala Leu Leu Val Val Asp Glu
65 70 75 80
Thr Leu Leu Glu Asn Asn Pro Thr Leu Leu Ala Val Asp Arg Ala Pro
85 90 95
Met Thr Tyr Ser Ile Asp Leu Pro Gly Leu Ala Ser Ser Asp Ser Phe
100 105 110
Leu Gln Val Glu Asp Pro Ser Asn Ser Ser Val Arg Gly Ala Val Asn
115 120 125
Asp Leu Leu Ala Lys Trp His Gln Asp Tyr Gly Gln Val Asn Asn Val
130 135 140
Pro Ala Arg Met Gln Tyr Glu Lys Ile Thr Ala His Ser Met Glu Gln
145 150 155 160
Leu Lys Val Lys Phe Gly Ser Asp Phe Glu Lys Thr Gly Asn Ser Leu
165 170 175
Asp Ile Asp Phe Asn Ser Val His Ser Gly Glu Lys Gln Ile Gln Ile
180 185 190
Val Asn Phe Lys Gln Ile Tyr Tyr Thr Val Ser Val Asp Ala Val Lys
195 200 205
Asn Pro Gly Asp Val Phe Gln Asp Thr Val Thr Val Glu Asp Leu Lys
210 215 220
Gln Arg Gly Ile Ser Ala Glu Arg Pro Leu Val Tyr Ile Ser Ser Val
225 230 235 240
Ala Tyr Gly Arg Gln Val Tyr Leu Lys Leu Glu Thr Thr Ser Lys Ser
245 250 255
Asp Glu Val Glu Ala Ala Phe Glu Ala Leu Ile Lys Gly Val Lys Val
260 265 270
Ala Pro Gln Thr Glu Trp Lys Gln Ile Leu Asp Asn Thr Glu Val Lys
275 280 285
Ala Val Ile Leu Cys Gly Asp Pro Ser Ser Gly Ala Arg Val Val Thr
290 295 300
Gly Lys Val Asp Met Val Glu Asp Leu Ile Gln Glu Gly Ser Arg Phe
305 310 315 320
Thr Ala Asp His Pro Gly Leu Pro Ile Ser Tyr Thr Thr Ser Phe Leu
325 330 335
Arg Asp Asn Val Val Ala Thr Phe Gln Asn Ser Thr Asp Tyr Val Glu
340 345 350
Thr Lys Val Thr Ala Tyr Arg Asn Gly Asp Leu Leu Leu Asp His Ser
355 360 365
Gly Ala Tyr Val Ala Gln Tyr Tyr Ile Thr Trp Asp Glu Leu Ser Tyr
370 375 380
Asp His Gln Gly Lys Glu Val Leu Thr Pro Lys Ala Trp Asp Arg Asn
385 390 395 400
Gly Gln Asp Leu Thr Ala His Phe Thr Thr Ser Ile Pro Leu Lys Gly
405 410 415
Asn Val Arg Asn Leu Ser Val Lys Ile Arg Glu Ala Thr Gly Leu Ala
420 425 430
Trp Glu Trp Trp Arg Thr Val Tyr Glu Lys Thr Asp Leu Pro Leu Val
435 440 445
Arg Lys Arg Thr Ile Ser Ile Trp Gly Thr Thr Leu Tyr Pro Gln Val
450 455 460
Glu Asp Lys Val Glu Asn Asp
465 470
<210> 3
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> rPly(T65C,G293C,C428A)引物CPUEM-A-F
<400> 3
taagaaggag atatacatat ggcgaacaaa gctgtgaacg atttc 45
<210> 4
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> rPly(T65C,G293C,C428A)引物CPUEM-A-R
<400> 4
cagtggtggt ggtggtggtg ctcgagttag tcgttctcaa ctttg 45
Claims (10)
1.一种重组肺炎球菌溶血素的纯化方法,其特征在于,其包括以下步骤:
(1)粗纯化:将精氨酸与表达所述重组肺炎球菌溶血素的菌体混合,获得粗产物;在粗纯化的体系中,所述精氨酸的终浓度为0.5~0.75M,所述菌体的终浓度为0.2~0.5g/mL,例如0.3g/mL;所述重组肺炎球菌溶血素的氨基酸序列如SEQ ID NO:2所示;
(2)精纯化:将所述粗产物经弱阴离子交换层析、疏水层析和强阴离子交换层析后,获得所述重组肺炎球菌溶血素;
所述弱阴离子交换层析的介质使用DEAE Sepharose FF,所述疏水层析的介质使用Phenyl Bestarose HP,所述强阴离子交换层析的介质使用QSepharose FF。
2.如权利要求1所述的纯化方法,其特征在于,步骤(1)包括:
(a)高渗处理:将菌体与精氨酸进行混合,进行搅拌;所述搅拌的时长为1~5小时例如3小时;
(b)低渗处理:使用稀释剂稀释,进行搅拌,获得稀释液;所述稀释剂例如为Tris-HCl,所述搅拌时长为12~20小时;
(c)超滤澄清:将步骤(c)中所述稀释液进行过滤,收集透过液;所述过滤使用的膜包孔径例如为0.22~0.45μm;
优选地,步骤(c)后还包括(d)超滤浓缩:将透过液进行超滤浓缩,收集截流液,将截流液使用置换液置换例如4次,获得粗制的重组肺炎球菌溶血素;所述置换液为Tris-HCl缓冲液;和/或,步骤(a)前还包括含有所述菌体的发酵液进行浓缩、过滤,获得浓缩的菌体;所述过滤使用的膜包孔径为0.22~0.45μm;
更优选地,步骤(a)中浓缩的菌体的浓度为0.3g/ml,所述精氨酸终浓度为0.5mol/L;步骤(b)中所述的稀释剂为50mM Tris-HCl、pH8.0,所述的稀释的倍数为10倍;和/或,步骤(c)中所述超滤浓缩使用的膜包孔径为10Kda~30Kda,所述的置换液为20mM Tris-HCl、pH8.0、含1mM EDTA和0.062M NaCl,和/或,所述的置换液和截流液的体积比为5:1。
3.如权利要求1所述的纯化方法,其特征在于,步骤(2)包括:
(2-1)将步骤(1)获得的粗产品上样于经平衡后的DEAE Sepharose FF,清洗、洗脱后收集洗脱液1;所述平衡、所述清洗和所述洗脱使用含NaCl的Tris-HCl缓冲液1,pH8.0-8.3优选8.0;所述平衡和所述清洗中使用的NaCl终浓度为0~0.1M;所述洗脱中使用的NaCl终浓度为0.1~0.2M优选0.12M;
(2-2)将上述步骤(2-1)中的洗脱液1与(NH4)2SO4混合获得混合液1,(NH4)2SO4终浓度为0.2~0.6M,将上述混合液上样于经平衡后的Phenyl Bestarose HP疏水层析柱,清洗、洗脱后收集洗脱液2;所述平衡和所述清洗使用含0.2~0.6M(NH4)2SO4的PB缓冲液;
(2-3)将上述步骤(2-2)中的洗脱液2稀释至电导率<5.0mS/cm后上样于经平衡后的QSepharose FF,清洗、洗脱后收集洗脱液3,所述稀释、所述平衡和所述清洗使用Tris-HCl缓冲液2,pH8.0-8.5优选pH8.5;所述洗脱使用含终浓度为0.04~0.13M NaCl的Tris-HCl缓冲液2;
优选地,步骤(2-1)中所述平衡和所述清洗使用20mM Tris-HCl、pH8.0、含1mM EDTA和0.062M NaCl,所述洗脱使用20mM Tris-HCl、pH8.0、含1mM EDTA和0.12M NaCl;
步骤(2-2)中所述平衡和所述清洗使用20mM PB、pH7.5、含1mM EDTA、0.6M(NH4)2SO4,所述洗脱使用20mM PB、pH7.5、含1mM EDTA;
步骤(2-3)中所述平衡、所述稀释和所述清洗使用100mM Tris-HCl、pH8.5、含1mMEDTA,所述洗脱使用100mM Tris-HCl、pH8.5、含1mM EDTA和0.12M NaCl。
4.如权利要求3所述的纯化方法,其特征在于,还包括:
(2-4)将上述步骤(2-3)中的洗脱液3进行超滤浓缩、置换,获得精制rPly;所述超滤浓缩使用的超滤膜包孔径为10Kda~30Kda;和/或,所述置换使用硼酸盐缓冲液;
优选地,所述置换使用含0.9%NaCl的200mM硼酸盐缓冲液、pH8.0。
5.如权利要求4所述的纯化方法,其特征在于,还包括:
(2-5)将上述步骤(2-4)中获得的精制的重组肺炎球菌溶血素过滤后,分装保存至温度2℃~﹣80℃;
优选地,所述过滤使用0.22μm滤器,和/或,所述分装保存温度为﹣80℃。
6.如权利要求3~5任一项所述的纯化方法,其特征在于,步骤(2-1)中所述平衡和所述清洗分别使用2倍柱体积和3倍柱体积的20mM Tris-HCl、pH8.0、含1mM EDTA和0.062MNaCl、所述洗脱使用3倍柱体积的20mM Tris-HCl、pH8.0、含1mM EDTA和0.12M NaCl;
步骤(2-2)中所述平衡和所述清洗分别使用2倍柱体积和3倍柱体积的20mM PB、pH7.5、含1mM EDTA、0.6M(NH4)2SO4、所述洗脱使用3倍柱体积的20mM PB、pH7.5、含1mM EDTA;
步骤(2-3)中所述平衡和所述清洗分别使用2倍柱体积和3倍柱体积的100mM Tris-HCl、pH8.5、含1mM EDTA、所述洗脱使用3倍柱体积的100mM Tris-HCl、pH8.5、含1mM EDTA和0.12M NaCl;
步骤(2-4)中所述置换使用10倍于洗脱液3的体积的200mM硼酸盐、pH8.0、含0.9%NaCl。
7.如权利要求1~6任一项所述的纯化方法,其特征在于,其包括以下步骤:
(I)培养表达氨基酸序列如SEQ ID NO:2所示的重组肺炎球菌溶血素的宿主细胞,所述宿主细胞为真核细胞或原核细胞;
(II)当OD600nm值例如为≥1,加入诱导剂进行诱导表达后即得;
优选地,所述重组肺炎球菌溶血素由核苷酸序列如SEQ ID NO:1所示的基因编码;
更优选地,步骤(I)中所述培养使用含有卡那霉素的三角瓶或发酵罐,步骤(II)中所述的诱导剂为IPTG;和/或,所述培养的温度为34~37℃,所述培养的pH为6.9~7.1,所述IPTG的浓度为0.1~1mM,所述诱导的温度为20~37℃,所述诱导的时间为2~6小时或过夜;
进一步更优选地,所述培养的温度为35℃,所述培养的pH为7.0,所述卡那霉素的终浓度为30μg/ml,所述IPTG的浓度为0.1mM或0.3mM,所述诱导的温度为32℃,所述诱导的时间为4小时。
8.如权利要求7所述的纯化方法,其特征在于,所述原核细胞为大肠杆菌;
优选地,所述大肠杆菌为BL21(DE3)菌株。
9.如权利要求7所述的纯化方法,其特征在于,所述宿主细胞包括表达载体,所述表达载体包含编码氨基酸序列如SEQ ID NO:2所示的重组肺炎球菌溶血素的核苷酸;
优选地,所述表达载体为原核表达载体或真核表达载体;
更优选地,所述原核表达载体为pET30a。
10.一种氨基酸序列如SEQ ID NO:2所示的重组肺炎球菌溶血素的保存方法,其特征在于,在-80±10℃条件下,使用含0.9%NaCl溶液的pH 8.0、0.2mol/L硼酸盐缓冲液保存所述重组肺炎球菌溶血素。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110896837.9A CN113480622A (zh) | 2021-08-05 | 2021-08-05 | 一种重组肺炎球菌溶血素制备及纯化的方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110896837.9A CN113480622A (zh) | 2021-08-05 | 2021-08-05 | 一种重组肺炎球菌溶血素制备及纯化的方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113480622A true CN113480622A (zh) | 2021-10-08 |
Family
ID=77944423
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110896837.9A Pending CN113480622A (zh) | 2021-08-05 | 2021-08-05 | 一种重组肺炎球菌溶血素制备及纯化的方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113480622A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114317486A (zh) * | 2021-12-30 | 2022-04-12 | 武汉赛维尔生物科技有限公司 | 一种末端脱氧核糖核苷酸转移酶TdT的纯化方法 |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1417226A (zh) * | 2001-11-09 | 2003-05-14 | 上海汉殷药业有限公司 | 从大肠杆菌中分离纯化重组硫氧还蛋白融合蛋白的方法及其应用 |
WO2012065262A1 (en) * | 2010-11-15 | 2012-05-24 | Sanofi Pasteur Limited | Expression, purification and refolding of recombinant chlamydia proteins, compositions and related methods |
CN102712680A (zh) * | 2008-12-24 | 2012-10-03 | 荷兰王国卫生福利和运动部国家公共卫生和环境研究所 | 修饰的肺炎链球菌溶血素(ply)多肽 |
CN105968182A (zh) * | 2016-06-03 | 2016-09-28 | 黄文林 | 一种重组人隐花色素蛋白I(hCRY1)的生产工艺及其组合物 |
CN107624070A (zh) * | 2015-05-04 | 2018-01-23 | 辉瑞大药厂 | B族链球菌多糖‑蛋白质缀合物、制造缀合物的方法、含缀合物的免疫原性组合物及其用途 |
WO2019077432A1 (en) * | 2017-10-16 | 2019-04-25 | Intas Pharmaceuticals Ltd. | IMPROVED PURIFICATION METHOD OF RECOMBINANT PTH (1-34) |
CN112661864A (zh) * | 2020-12-30 | 2021-04-16 | 华润昂德生物药业有限公司 | 重组人GLP-1-Fc融合蛋白的纯化方法 |
-
2021
- 2021-08-05 CN CN202110896837.9A patent/CN113480622A/zh active Pending
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1417226A (zh) * | 2001-11-09 | 2003-05-14 | 上海汉殷药业有限公司 | 从大肠杆菌中分离纯化重组硫氧还蛋白融合蛋白的方法及其应用 |
CN102712680A (zh) * | 2008-12-24 | 2012-10-03 | 荷兰王国卫生福利和运动部国家公共卫生和环境研究所 | 修饰的肺炎链球菌溶血素(ply)多肽 |
CN106008679A (zh) * | 2008-12-24 | 2016-10-12 | 荷兰王国卫生福利和运动部国家公共卫生和环境研究所 | 修饰的肺炎链球菌溶血素(ply)多肽 |
WO2012065262A1 (en) * | 2010-11-15 | 2012-05-24 | Sanofi Pasteur Limited | Expression, purification and refolding of recombinant chlamydia proteins, compositions and related methods |
CN107624070A (zh) * | 2015-05-04 | 2018-01-23 | 辉瑞大药厂 | B族链球菌多糖‑蛋白质缀合物、制造缀合物的方法、含缀合物的免疫原性组合物及其用途 |
CN105968182A (zh) * | 2016-06-03 | 2016-09-28 | 黄文林 | 一种重组人隐花色素蛋白I(hCRY1)的生产工艺及其组合物 |
WO2019077432A1 (en) * | 2017-10-16 | 2019-04-25 | Intas Pharmaceuticals Ltd. | IMPROVED PURIFICATION METHOD OF RECOMBINANT PTH (1-34) |
CN112661864A (zh) * | 2020-12-30 | 2021-04-16 | 华润昂德生物药业有限公司 | 重组人GLP-1-Fc融合蛋白的纯化方法 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114317486A (zh) * | 2021-12-30 | 2022-04-12 | 武汉赛维尔生物科技有限公司 | 一种末端脱氧核糖核苷酸转移酶TdT的纯化方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP2560738B1 (en) | Simple method for simultaneous removal of multiple impurities from culture supernatants to ultralow levels | |
US20100286373A1 (en) | Immunoglobulin affinity ligand | |
KR101831300B1 (ko) | 재조합 대장균으로부터 인간 과립구 콜로니 자극인자를 정제하는 방법 | |
MX2013014773A (es) | Procedimiento de purificacion de formas nativas o mutantes de la toxina difterica. | |
JPS6269998A (ja) | 不溶性封入体からの蛋白質の精製および活性化 | |
US8536302B2 (en) | Dockerin polypeptide and method of purifying recombinant fused protein using the same | |
CN113150120B (zh) | 发酵液中猪肌红蛋白的分离纯化方法 | |
WO2005068489A1 (ja) | 2価陽イオン存在下加熱処理によるヒト血清アルブミンの製造方法 | |
CN109929027B (zh) | 采用线性洗脱步骤的重组融合蛋白纯化方法 | |
CN113480622A (zh) | 一种重组肺炎球菌溶血素制备及纯化的方法 | |
CN112898387B (zh) | 一种重组金黄色葡萄球菌疫苗抗原蛋白的纯化方法 | |
EP0446582B1 (en) | Method for producing of recombinant human cysteineless gamma-interferon free of methionine at n-terminal | |
JP4088584B2 (ja) | 融合タンパク質から目的タンパク質を分離する方法。 | |
CN111676261A (zh) | 一种高纯度重组白介素-2的制备工艺 | |
WO2019143193A1 (ko) | 재조합 폴리펩타이드 생산용 n-말단 융합 파트너 및 이를 이용하여 재조합 폴리 펩타이드를 생산하는방법 | |
CN109776654B (zh) | 一种亲和肽及其应用 | |
CN113069538B (zh) | 一种制备重组金黄色葡萄球菌疫苗的方法 | |
CN110563832A (zh) | 一种高纯度重组促卵泡刺激素纯化方法 | |
CN116217680B (zh) | 碱稳定性高的免疫球蛋白结合蛋白及其应用 | |
García-Orozco et al. | Recombinant bacterial expression of the lysozyme from the tobacco-hornworm Manduca sexta with activity at low temperatures | |
CN113072639B (zh) | 一种高纯度重组水蛭素的纯化方法 | |
CN116410295A (zh) | 一种大肠杆菌表达物的纯化方法 | |
CN104945488B (zh) | 一种具有免疫球蛋白结合能力的多肽 | |
CN113321714B (zh) | SARS-CoV-2的重组N蛋白及其制备与纯化方法 | |
WO2023093826A1 (zh) | 一种蛋白的生产方法及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20211008 |
|
WD01 | Invention patent application deemed withdrawn after publication |