CN113667022A - Fusion protein and application thereof - Google Patents
Fusion protein and application thereof Download PDFInfo
- Publication number
- CN113667022A CN113667022A CN202110984130.3A CN202110984130A CN113667022A CN 113667022 A CN113667022 A CN 113667022A CN 202110984130 A CN202110984130 A CN 202110984130A CN 113667022 A CN113667022 A CN 113667022A
- Authority
- CN
- China
- Prior art keywords
- ala
- hepatitis
- protein
- gly
- amino acid
- 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
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 29
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 29
- 241000711549 Hepacivirus C Species 0.000 claims abstract description 25
- 108700039791 Hepatitis C virus nucleocapsid Proteins 0.000 claims abstract description 19
- 230000014509 gene expression Effects 0.000 claims abstract description 13
- 208000005176 Hepatitis C Diseases 0.000 claims abstract description 12
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 12
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 12
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 12
- 101800000515 Non-structural protein 3 Proteins 0.000 claims abstract description 11
- 244000005700 microbiome Species 0.000 claims abstract description 9
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 25
- 108090000623 proteins and genes Proteins 0.000 claims description 25
- 102000004169 proteins and genes Human genes 0.000 claims description 23
- 108020004414 DNA Proteins 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 102000053602 DNA Human genes 0.000 claims description 6
- 108010075254 C-Peptide Proteins 0.000 claims description 5
- 210000004899 c-terminal region Anatomy 0.000 claims description 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 3
- 101710132601 Capsid protein Proteins 0.000 claims description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 2
- 229930182817 methionine Natural products 0.000 claims description 2
- 210000002966 serum Anatomy 0.000 abstract description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 38
- 238000001514 detection method Methods 0.000 description 22
- 239000000243 solution Substances 0.000 description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 16
- 238000003756 stirring Methods 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- 238000001179 sorption measurement Methods 0.000 description 12
- 239000012528 membrane Substances 0.000 description 11
- 238000003908 quality control method Methods 0.000 description 11
- 239000007788 liquid Substances 0.000 description 9
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 8
- 238000007789 sealing Methods 0.000 description 8
- 101710144111 Non-structural protein 3 Proteins 0.000 description 7
- 239000000427 antigen Substances 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 238000005520 cutting process Methods 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 238000001035 drying Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 108010061238 threonyl-glycine Proteins 0.000 description 6
- 229920000936 Agarose Polymers 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 108010047495 alanylglycine Proteins 0.000 description 5
- 239000011248 coating agent Substances 0.000 description 5
- 238000000576 coating method Methods 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000002791 soaking Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- RLMISHABBKUNFO-WHFBIAKZSA-N Ala-Ala-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O RLMISHABBKUNFO-WHFBIAKZSA-N 0.000 description 4
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 4
- 108010087924 alanylproline Proteins 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000001976 enzyme digestion Methods 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 239000008213 purified water Substances 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- FKJQNJCQTKUBCD-XPUUQOCRSA-N Gly-Ala-His Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)O FKJQNJCQTKUBCD-XPUUQOCRSA-N 0.000 description 3
- 108010079364 N-glycylalanine Proteins 0.000 description 3
- 239000000020 Nitrocellulose Substances 0.000 description 3
- 101150038760 Ns3 gene Proteins 0.000 description 3
- KIZQGKLMXKGDIV-BQBZGAKWSA-N Pro-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 KIZQGKLMXKGDIV-BQBZGAKWSA-N 0.000 description 3
- CLNJSLSHKJECME-BQBZGAKWSA-N Pro-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H]1CCCN1 CLNJSLSHKJECME-BQBZGAKWSA-N 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 230000002335 preservative effect Effects 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 2
- XVZCXCTYGHPNEM-IHRRRGAJSA-N (2s)-1-[(2s)-2-[[(2s)-2-amino-4-methylpentanoyl]amino]-4-methylpentanoyl]pyrrolidine-2-carboxylic acid Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(O)=O XVZCXCTYGHPNEM-IHRRRGAJSA-N 0.000 description 2
- QMOQBVOBWVNSNO-UHFFFAOYSA-N 2-[[2-[[2-[(2-azaniumylacetyl)amino]acetyl]amino]acetyl]amino]acetate Chemical compound NCC(=O)NCC(=O)NCC(=O)NCC(O)=O QMOQBVOBWVNSNO-UHFFFAOYSA-N 0.000 description 2
- CXRCVCURMBFFOL-FXQIFTODSA-N Ala-Ala-Pro Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O CXRCVCURMBFFOL-FXQIFTODSA-N 0.000 description 2
- ZVFVBBGVOILKPO-WHFBIAKZSA-N Ala-Gly-Ala Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O ZVFVBBGVOILKPO-WHFBIAKZSA-N 0.000 description 2
- ADSGHMXEAZJJNF-DCAQKATOSA-N Ala-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N ADSGHMXEAZJJNF-DCAQKATOSA-N 0.000 description 2
- RTZCUEHYUQZIDE-WHFBIAKZSA-N Ala-Ser-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RTZCUEHYUQZIDE-WHFBIAKZSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 206010016654 Fibrosis Diseases 0.000 description 2
- LHYJCVCQPWRMKZ-WEDXCCLWSA-N Gly-Leu-Thr Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LHYJCVCQPWRMKZ-WEDXCCLWSA-N 0.000 description 2
- HHRODZSXDXMUHS-LURJTMIESA-N Gly-Met-Gly Chemical compound CSCC[C@H](NC(=O)C[NH3+])C(=O)NCC([O-])=O HHRODZSXDXMUHS-LURJTMIESA-N 0.000 description 2
- GGLIDLCEPDHEJO-BQBZGAKWSA-N Gly-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)CN GGLIDLCEPDHEJO-BQBZGAKWSA-N 0.000 description 2
- 241000880493 Leptailurus serval Species 0.000 description 2
- XVZCXCTYGHPNEM-UHFFFAOYSA-N Leu-Leu-Pro Natural products CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(O)=O XVZCXCTYGHPNEM-UHFFFAOYSA-N 0.000 description 2
- VTFXTWDFPTWNJY-RHYQMDGZSA-N Pro-Leu-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VTFXTWDFPTWNJY-RHYQMDGZSA-N 0.000 description 2
- AIOWVDNPESPXRB-YTWAJWBKSA-N Pro-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2)O AIOWVDNPESPXRB-YTWAJWBKSA-N 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 2
- 239000000853 adhesive Substances 0.000 description 2
- 230000001070 adhesive effect Effects 0.000 description 2
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 2
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 238000013329 compounding Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000006806 disease prevention Effects 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 108010001064 glycyl-glycyl-glycyl-glycine Proteins 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 229940127121 immunoconjugate Drugs 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 108010090894 prolylleucine Proteins 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- 229940038773 trisodium citrate Drugs 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 1
- LJFNNUBZSZCZFN-WHFBIAKZSA-N Ala-Gly-Cys Chemical compound N[C@@H](C)C(=O)NCC(=O)N[C@@H](CS)C(=O)O LJFNNUBZSZCZFN-WHFBIAKZSA-N 0.000 description 1
- VGPWRRFOPXVGOH-BYPYZUCNSA-N Ala-Gly-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)NCC(O)=O VGPWRRFOPXVGOH-BYPYZUCNSA-N 0.000 description 1
- CWEAKSWWKHGTRJ-BQBZGAKWSA-N Ala-Gly-Met Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O CWEAKSWWKHGTRJ-BQBZGAKWSA-N 0.000 description 1
- SMCGQGDVTPFXKB-XPUUQOCRSA-N Ala-Gly-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N SMCGQGDVTPFXKB-XPUUQOCRSA-N 0.000 description 1
- OKIKVSXTXVVFDV-MMWGEVLESA-N Ala-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C)N OKIKVSXTXVVFDV-MMWGEVLESA-N 0.000 description 1
- MNZHHDPWDWQJCQ-YUMQZZPRSA-N Ala-Leu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O MNZHHDPWDWQJCQ-YUMQZZPRSA-N 0.000 description 1
- MEFILNJXAVSUTO-JXUBOQSCSA-N Ala-Leu-Thr Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MEFILNJXAVSUTO-JXUBOQSCSA-N 0.000 description 1
- XWFWAXPOLRTDFZ-FXQIFTODSA-N Ala-Pro-Ser Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O XWFWAXPOLRTDFZ-FXQIFTODSA-N 0.000 description 1
- NCQMBSJGJMYKCK-ZLUOBGJFSA-N Ala-Ser-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O NCQMBSJGJMYKCK-ZLUOBGJFSA-N 0.000 description 1
- ARHJJAAWNWOACN-FXQIFTODSA-N Ala-Ser-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O ARHJJAAWNWOACN-FXQIFTODSA-N 0.000 description 1
- KUFVXLQLDHJVOG-SHGPDSBTSA-N Ala-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C)N)O KUFVXLQLDHJVOG-SHGPDSBTSA-N 0.000 description 1
- VHAQSYHSDKERBS-XPUUQOCRSA-N Ala-Val-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O VHAQSYHSDKERBS-XPUUQOCRSA-N 0.000 description 1
- REWSWYIDQIELBE-FXQIFTODSA-N Ala-Val-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O REWSWYIDQIELBE-FXQIFTODSA-N 0.000 description 1
- NRVQLLDIJJEIIZ-VZFHVOOUSA-N Cys-Thr-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](CS)N)O NRVQLLDIJJEIIZ-VZFHVOOUSA-N 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- PYTZFYUXZZHOAD-WHFBIAKZSA-N Gly-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)CN PYTZFYUXZZHOAD-WHFBIAKZSA-N 0.000 description 1
- UGVQELHRNUDMAA-BYPYZUCNSA-N Gly-Ala-Gly Chemical compound [NH3+]CC(=O)N[C@@H](C)C(=O)NCC([O-])=O UGVQELHRNUDMAA-BYPYZUCNSA-N 0.000 description 1
- QXPRJQPCFXMCIY-NKWVEPMBSA-N Gly-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN QXPRJQPCFXMCIY-NKWVEPMBSA-N 0.000 description 1
- QSDKBRMVXSWAQE-BFHQHQDPSA-N Gly-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN QSDKBRMVXSWAQE-BFHQHQDPSA-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
- CCQOOWAONKGYKQ-BYPYZUCNSA-N Gly-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)CN CCQOOWAONKGYKQ-BYPYZUCNSA-N 0.000 description 1
- OLPPXYMMIARYAL-QMMMGPOBSA-N Gly-Gly-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)CN OLPPXYMMIARYAL-QMMMGPOBSA-N 0.000 description 1
- HMHRTKOWRUPPNU-RCOVLWMOSA-N Gly-Ile-Gly Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O HMHRTKOWRUPPNU-RCOVLWMOSA-N 0.000 description 1
- BHPQOIPBLYJNAW-NGZCFLSTSA-N Gly-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN BHPQOIPBLYJNAW-NGZCFLSTSA-N 0.000 description 1
- LXTRSHQLGYINON-DTWKUNHWSA-N Gly-Met-Pro Chemical compound CSCC[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN LXTRSHQLGYINON-DTWKUNHWSA-N 0.000 description 1
- NSVOVKWEKGEOQB-LURJTMIESA-N Gly-Pro-Gly Chemical compound NCC(=O)N1CCC[C@H]1C(=O)NCC(O)=O NSVOVKWEKGEOQB-LURJTMIESA-N 0.000 description 1
- WNGHUXFWEWTKAO-YUMQZZPRSA-N Gly-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN WNGHUXFWEWTKAO-YUMQZZPRSA-N 0.000 description 1
- TVTZEOHWHUVYCG-KYNKHSRBSA-N Gly-Thr-Thr Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O TVTZEOHWHUVYCG-KYNKHSRBSA-N 0.000 description 1
- GWCJMBNBFYBQCV-XPUUQOCRSA-N Gly-Val-Ala Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O GWCJMBNBFYBQCV-XPUUQOCRSA-N 0.000 description 1
- AFMOTCMSEBITOE-YEPSODPASA-N Gly-Val-Thr Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O AFMOTCMSEBITOE-YEPSODPASA-N 0.000 description 1
- 206010019663 Hepatic failure Diseases 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- XINDHUAGVGCNSF-QSFUFRPTSA-N His-Ala-Ile Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O XINDHUAGVGCNSF-QSFUFRPTSA-N 0.000 description 1
- VCBWXASUBZIFLQ-IHRRRGAJSA-N His-Pro-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O VCBWXASUBZIFLQ-IHRRRGAJSA-N 0.000 description 1
- NKVZTQVGUNLLQW-JBDRJPRFSA-N Ile-Ala-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)O)N NKVZTQVGUNLLQW-JBDRJPRFSA-N 0.000 description 1
- CISBRYJZMFWOHJ-JBDRJPRFSA-N Ile-Ala-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CS)C(=O)O)N CISBRYJZMFWOHJ-JBDRJPRFSA-N 0.000 description 1
- DFFTXLCCDFYRKD-MBLNEYKQSA-N Ile-Gly-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)O)N DFFTXLCCDFYRKD-MBLNEYKQSA-N 0.000 description 1
- FZWVCYCYWCLQDH-NHCYSSNCSA-N Ile-Leu-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)O)N FZWVCYCYWCLQDH-NHCYSSNCSA-N 0.000 description 1
- IVXJIMGDOYRLQU-XUXIUFHCSA-N Ile-Pro-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O IVXJIMGDOYRLQU-XUXIUFHCSA-N 0.000 description 1
- CNMOKANDJMLAIF-CIQUZCHMSA-N Ile-Thr-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O CNMOKANDJMLAIF-CIQUZCHMSA-N 0.000 description 1
- KXUKTDGKLAOCQK-LSJOCFKGSA-N Ile-Val-Gly Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O KXUKTDGKLAOCQK-LSJOCFKGSA-N 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- OXRLYTYUXAQTHP-YUMQZZPRSA-N Leu-Gly-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(O)=O OXRLYTYUXAQTHP-YUMQZZPRSA-N 0.000 description 1
- YFBBUHJJUXXZOF-UWVGGRQHSA-N Leu-Gly-Pro Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N1CCC[C@H]1C(O)=O YFBBUHJJUXXZOF-UWVGGRQHSA-N 0.000 description 1
- HYMLKESRWLZDBR-WEDXCCLWSA-N Leu-Gly-Thr Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O HYMLKESRWLZDBR-WEDXCCLWSA-N 0.000 description 1
- DBSLVQBXKVKDKJ-BJDJZHNGSA-N Leu-Ile-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O DBSLVQBXKVKDKJ-BJDJZHNGSA-N 0.000 description 1
- RXGLHDWAZQECBI-SRVKXCTJSA-N Leu-Leu-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O RXGLHDWAZQECBI-SRVKXCTJSA-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
- FLNPJLDPGMLWAU-UWVGGRQHSA-N Leu-Met-Gly Chemical compound OC(=O)CNC(=O)[C@H](CCSC)NC(=O)[C@@H](N)CC(C)C FLNPJLDPGMLWAU-UWVGGRQHSA-N 0.000 description 1
- JVTYXRRFZCEPPK-RHYQMDGZSA-N Leu-Met-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CC(C)C)N)O JVTYXRRFZCEPPK-RHYQMDGZSA-N 0.000 description 1
- DPURXCQCHSQPAN-AVGNSLFASA-N Leu-Pro-Pro Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DPURXCQCHSQPAN-AVGNSLFASA-N 0.000 description 1
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-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
- FGZVGOAAROXFAB-IXOXFDKPSA-N Leu-Thr-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CC(C)C)N)O FGZVGOAAROXFAB-IXOXFDKPSA-N 0.000 description 1
- LJBVRCDPWOJOEK-PPCPHDFISA-N Leu-Thr-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LJBVRCDPWOJOEK-PPCPHDFISA-N 0.000 description 1
- QWWPYKKLXWOITQ-VOAKCMCISA-N Leu-Thr-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(C)C QWWPYKKLXWOITQ-VOAKCMCISA-N 0.000 description 1
- AAKRWBIIGKPOKQ-ONGXEEELSA-N Leu-Val-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O AAKRWBIIGKPOKQ-ONGXEEELSA-N 0.000 description 1
- QRHWTCJBCLGYRB-FXQIFTODSA-N Met-Ala-Cys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CS)C(O)=O QRHWTCJBCLGYRB-FXQIFTODSA-N 0.000 description 1
- CIDICGYKRUTYLE-FXQIFTODSA-N Met-Ser-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O CIDICGYKRUTYLE-FXQIFTODSA-N 0.000 description 1
- DBMLDOWSVHMQQN-XGEHTFHBSA-N Met-Ser-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DBMLDOWSVHMQQN-XGEHTFHBSA-N 0.000 description 1
- IFMDQWDAJUMMJC-DCAQKATOSA-N Pro-Ala-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O IFMDQWDAJUMMJC-DCAQKATOSA-N 0.000 description 1
- HAAQQNHQZBOWFO-LURJTMIESA-N Pro-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H]1CCCN1 HAAQQNHQZBOWFO-LURJTMIESA-N 0.000 description 1
- KIDXAAQVMNLJFQ-KZVJFYERSA-N Pro-Thr-Ala Chemical compound C[C@@H](O)[C@H](NC(=O)[C@@H]1CCCN1)C(=O)N[C@@H](C)C(O)=O KIDXAAQVMNLJFQ-KZVJFYERSA-N 0.000 description 1
- FDMCIBSQRKFSTJ-RHYQMDGZSA-N Pro-Thr-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O FDMCIBSQRKFSTJ-RHYQMDGZSA-N 0.000 description 1
- YDTUEBLEAVANFH-RCWTZXSCSA-N Pro-Val-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 YDTUEBLEAVANFH-RCWTZXSCSA-N 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- BRKHVZNDAOMAHX-BIIVOSGPSA-N Ser-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N BRKHVZNDAOMAHX-BIIVOSGPSA-N 0.000 description 1
- YMTLKLXDFCSCNX-BYPYZUCNSA-N Ser-Gly-Gly Chemical compound OC[C@H](N)C(=O)NCC(=O)NCC(O)=O YMTLKLXDFCSCNX-BYPYZUCNSA-N 0.000 description 1
- SFTZWNJFZYOLBD-ZDLURKLDSA-N Ser-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO SFTZWNJFZYOLBD-ZDLURKLDSA-N 0.000 description 1
- FUMGHWDRRFCKEP-CIUDSAMLSA-N Ser-Leu-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O FUMGHWDRRFCKEP-CIUDSAMLSA-N 0.000 description 1
- VGQVAVQWKJLIRM-FXQIFTODSA-N Ser-Ser-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O VGQVAVQWKJLIRM-FXQIFTODSA-N 0.000 description 1
- PURRNJBBXDDWLX-ZDLURKLDSA-N Ser-Thr-Gly Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CO)N)O PURRNJBBXDDWLX-ZDLURKLDSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical group 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 1
- TYVAWPFQYFPSBR-BFHQHQDPSA-N Thr-Ala-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)NCC(O)=O TYVAWPFQYFPSBR-BFHQHQDPSA-N 0.000 description 1
- BSNZTJXVDOINSR-JXUBOQSCSA-N Thr-Ala-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O BSNZTJXVDOINSR-JXUBOQSCSA-N 0.000 description 1
- DWYAUVCQDTZIJI-VZFHVOOUSA-N Thr-Ala-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O DWYAUVCQDTZIJI-VZFHVOOUSA-N 0.000 description 1
- DSLHSTIUAPKERR-XGEHTFHBSA-N Thr-Cys-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(O)=O DSLHSTIUAPKERR-XGEHTFHBSA-N 0.000 description 1
- SLUWOCTZVGMURC-BFHQHQDPSA-N Thr-Gly-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O SLUWOCTZVGMURC-BFHQHQDPSA-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
- DJDSEDOKJTZBAR-ZDLURKLDSA-N Thr-Gly-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O DJDSEDOKJTZBAR-ZDLURKLDSA-N 0.000 description 1
- KBBRNEDOYWMIJP-KYNKHSRBSA-N Thr-Gly-Thr Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)O)N)O KBBRNEDOYWMIJP-KYNKHSRBSA-N 0.000 description 1
- YJCVECXVYHZOBK-KNZXXDILSA-N Thr-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H]([C@@H](C)O)N YJCVECXVYHZOBK-KNZXXDILSA-N 0.000 description 1
- ODXKUIGEPAGKKV-KATARQTJSA-N Thr-Leu-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)O)N)O ODXKUIGEPAGKKV-KATARQTJSA-N 0.000 description 1
- XIULAFZYEKSGAJ-IXOXFDKPSA-N Thr-Leu-His Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CNC=N1 XIULAFZYEKSGAJ-IXOXFDKPSA-N 0.000 description 1
- GVMXJJAJLIEASL-ZJDVBMNYSA-N Thr-Pro-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O GVMXJJAJLIEASL-ZJDVBMNYSA-N 0.000 description 1
- SGAOHNPSEPVAFP-ZDLURKLDSA-N Thr-Ser-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SGAOHNPSEPVAFP-ZDLURKLDSA-N 0.000 description 1
- NDZYTIMDOZMECO-SHGPDSBTSA-N Thr-Thr-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O NDZYTIMDOZMECO-SHGPDSBTSA-N 0.000 description 1
- PJCYRZVSACOYSN-ZJDVBMNYSA-N Thr-Thr-Met Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(O)=O PJCYRZVSACOYSN-ZJDVBMNYSA-N 0.000 description 1
- BKVICMPZWRNWOC-RHYQMDGZSA-N Thr-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)[C@@H](C)O BKVICMPZWRNWOC-RHYQMDGZSA-N 0.000 description 1
- ZLFHAAGHGQBQQN-AEJSXWLSSA-N Val-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C(C)C)N ZLFHAAGHGQBQQN-AEJSXWLSSA-N 0.000 description 1
- ZLFHAAGHGQBQQN-GUBZILKMSA-N Val-Ala-Pro Natural products CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O ZLFHAAGHGQBQQN-GUBZILKMSA-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
- CWSIBTLMMQLPPZ-FXQIFTODSA-N Val-Cys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](C(C)C)N CWSIBTLMMQLPPZ-FXQIFTODSA-N 0.000 description 1
- BMOFUVHDBROBSE-DCAQKATOSA-N Val-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](C(C)C)N BMOFUVHDBROBSE-DCAQKATOSA-N 0.000 description 1
- MIKHIIQMRFYVOR-RCWTZXSCSA-N Val-Pro-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C(C)C)N)O MIKHIIQMRFYVOR-RCWTZXSCSA-N 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000001467 acupuncture Methods 0.000 description 1
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 108010000434 glycyl-alanyl-leucine Proteins 0.000 description 1
- 108010078326 glycyl-glycyl-valine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 208000010710 hepatitis C virus infection Diseases 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 108010036413 histidylglycine Proteins 0.000 description 1
- 108010025306 histidylleucine Proteins 0.000 description 1
- 108010085325 histidylproline Proteins 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 108010031424 isoleucyl-prolyl-proline Proteins 0.000 description 1
- 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 description 1
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 1
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 231100000835 liver failure Toxicity 0.000 description 1
- 208000007903 liver failure Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 101150106875 malE gene Proteins 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000003761 preservation solution Substances 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 108010020755 prolyl-glycyl-glycine Proteins 0.000 description 1
- 108700042769 prolyl-leucyl-glycine Proteins 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000011265 semifinished product Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000012475 sodium chloride buffer Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical group [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 229940033663 thimerosal Drugs 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 229940074410 trehalose Drugs 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
- 238000005406 washing Methods 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
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/24—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a MBP (maltose binding protein)-tag
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
- C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pathology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Peptides Or Proteins (AREA)
- Virology (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Communicable Diseases (AREA)
Abstract
The invention provides any of the following products: (B1) a fusion protein is composed of hepatitis C virus core protein and hepatitis C virus non-structural protein NS 3; (B2) a nucleic acid molecule encoding the fusion protein; (B3) an expression cassette comprising the nucleic acid molecule of (B2); (B4) a recombinant vector comprising (B2) the nucleic acid molecule or a recombinant vector comprising (B3) the expression cassette; (B5) a recombinant microorganism containing (B2) the nucleic acid molecule, or a recombinant microorganism containing (B3) the expression cassette, or a recombinant microorganism containing B4) the recombinant vector. The fusion protein can be combined with an antibody in serum of a hepatitis C patient.
Description
Technical Field
The invention relates to the field of biological medicine, and particularly relates to a fusion protein and application thereof.
Background
Viral hepatitis C, abbreviated as hepatitis C and hepatitis C, is a viral hepatitis caused by Hepatitis C Virus (HCV) infection, and is mainly transmitted by blood transfusion, acupuncture, drug inhalation and the like, according to the statistics of the world health organization, the global HCV infection rate is about 3%, about 1.8 hundred million people are estimated to be infected with HCV, and about 3.5 ten thousand cases of new hepatitis C are generated every year. Hepatitis c is a global epidemic that can lead to chronic inflammatory necrosis and fibrosis of the liver, and some patients can develop cirrhosis and even liver cancer. The mortality rate associated with HCV infection (death from liver failure and hepatocellular carcinoma) will continue to increase over the next 20 years, posing significant health and life risks to the patient and becoming a serious social and public health problem. The host may produce antibodies to only one antigen or antibodies to multiple antigens, with differences in the production of antibodies to HCV at different stages of HCV infection and under different physiological conditions. Therefore, the detection reagent for anti-HCV contains a plurality of antigens, and thus the detection rate can be improved. In addition, the HCV recombinant antigen is used as the main component of the HCV antibody diagnostic reagent and plays a decisive role in the quality and the cost of the HCV antibody detection kit.
Disclosure of Invention
The invention solves the technical problem of how to detect hepatitis C.
The invention provides any of the following products:
(B1) a fusion protein is composed of hepatitis C virus core protein and hepatitis C virus non-structural protein NS 3;
(B2) a nucleic acid molecule encoding the fusion protein;
(B3) an expression cassette comprising the nucleic acid molecule of (B2);
(B4) a recombinant vector comprising (B2) the nucleic acid molecule or a recombinant vector comprising (B3) the expression cassette;
(B5) a recombinant microorganism containing (B2) the nucleic acid molecule, or a recombinant microorganism containing (B3) the expression cassette, or a recombinant microorganism containing B4) the recombinant vector.
Alternatively, the hepatitis c virus core protein and the hepatitis c virus non-structural protein NS3 are linked by a linker peptide.
Optionally, the amino acid sequence of the fusion protein is composed of the amino acid sequence of the hepatitis C virus core protein, the amino acid sequence of the connecting peptide and the amino acid sequence of the hepatitis C virus non-structural protein NS3 in sequence from the N terminal to the C terminal;
or the like, or, alternatively,
the amino acid sequence of the fusion protein consists of an amino acid sequence of a tag protein, methionine, an amino acid sequence of a core protein of the hepatitis C virus, an amino acid sequence of a connecting peptide and an amino acid sequence of a non-structural protein NS3 of the hepatitis C virus in sequence from the N terminal to the C terminal.
Optionally, the amino acid sequence of the hepatitis C virus core protein is SEQ ID No.2, 2-172; the nucleotide sequence of the coding DNA molecule is SEQ ID No.1(5 '-3') 4-516;
the amino acid sequence of the hepatitis C virus non-structural protein NS3 is 182-555 bit of SEQ ID No. 2; the nucleotide sequence of the coding DNA molecule is SEQ ID No.1(5 '-3') at position 544-1665;
the connecting peptide has the amino acid sequence of 173-181 of SEQ ID No. 2; the nucleotide sequence of the coding DNA molecule is 517-543 th site of SEQ ID No.1(5 '-3');
the tag protein is an MBP tag.
The application of the fusion protein in detecting hepatitis C; optionally, the sample to be tested is serum.
The technical scheme of the invention has the following advantages:
1. the antigen prepared by the method can be combined with an antibody in serum of a hepatitis C patient.
The vector is used for expressing HCV Core/NS3 recombinant fusion protein by a pMAL-C2 vector, the vector is provided with an MBP label, the MBP label is expressed at the N end of a target protein in a fusion manner, a TEV enzyme cutting site is connected behind the MBP label through PCR, and the MBP label is convenient to cut after expression. After the construction is successful, the expression of the target protein is expressed and purified. After expression, the MBP label is tried to be cut off, but the protein is unstable and easy to precipitate after the label is cut off, and the solubility of the protein is increased due to the existence of the MBP label. Thus, the protein with the label is labeled and detected by colloidal gold, and the existence of the MBP label does not influence the sensitivity and specificity of the detection reagent. While the skilled artisan will typically cleave the MBP tag, it has been unexpectedly discovered that the protein is more stable without cleavage and does not affect the sensitivity and specificity of the detection reagents.
Note: MBP (maltose binding protein) tag protein is 40kDa in size and is encoded by the malE gene of E.coli K12.
2. The invention provides a kit for specifically detecting hepatitis C, wherein fusion protein is used as an antigen, and can sensitively and specifically detect hepatitis C.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the results of agarose gel electrophoresis in example 1; in the figure, Marker M is Marker, and markers 1 and 2 are amplified HCV Core/NS3 genes;
FIG. 2 shows the results of protein detection by SDS-PAGE in example 1; reference numerals 1 to 5 are five replicates;
FIG. 3 shows the results of testing a portion of negative specimens in example 3;
FIG. 4 the results of the detection of a portion of the positive specimens in example 3;
FIG. 5 is a schematic view of a colloidal gold test strip for detecting Hepatitis C Virus (HCV) antibodies in example 2 of the present invention;
1-substrate, 2-loading pad, 3-colloidal gold adsorption pad, 4-bearing film, 5-water absorption pad, 6-protective film, T-detection line T, C-quality control line C.
Detailed Description
EXAMPLE 1 acquisition of HCV Core/NS3 fusion protein
1.1 biosynthesis of HCV Core/NS3 gene sequence with nucleotide sequence as SEQ ID NO.2, wherein the 1 st to 172 nd position is HCV Core gene sequence, the 183 rd and 555 nd position is NS3 gene sequence, and the 173 th to 182 nd position is linker gene sequence. The amino acid sequence of the protein coded by the HCV Core/NS3 gene is shown as SEQ ID NO. 1.
1.2 primer sequences: upstream primer ccggaattcatgagcaccaatccgaaa (SEQ ID NO.3) and downstream primer: cccaagcttttagcattcctccatttcatc (SEQ ID NO.4) SEQ ID NO.1 was amplified, restriction sites EcoR I/Hind III were added, and the PCR system was as follows:
template 1ul, 5 'end primer 1ul, 3' end primer 1ul, dNTP 4ul, Mg2+buffer 5ul、Mg2+4ul, Taq 1ul, water make up to 50 ul.
The PCR amplification procedure was:
1.3 agarose gel electrophoresis to recover the PCR product. The results of agarose gel electrophoresis are shown in FIG. 1.
1.4 enzyme digestion
PCR product obtained by enzyme digestion 1.3 and pMAl-C2The vector and the enzyme digestion system are as follows:
1.5 purification
And (3) carrying out electrophoresis on the enzyme digestion product for 15min at a constant voltage of 220V under the action of 0.8% agarose voltage, quickly cutting off the agarose block containing the target DNA by using a clean blade under an ultraviolet lamp, adding the agarose block into a 1.5ml centrifuge tube, weighing the weight of the cut agarose block by using an electronic balance, and purifying.
The purification (Omega: Plasmid Mini Kit I) was carried out as follows:
(1) adding a corresponding amount of Buffer I into the gel block according to the calculation of 1mg to 1ul, mixing, and placing in a 56 ℃ water bath kettle to completely melt the agarose block;
(2) transferring the solution into Spin Column, centrifuging at 10000rpm for 1min, and removing the filtrate;
(3) adding 300ul Binding Buffer, centrifuging at 10000rpm for 1 min;
(4) adding 700ul of SPW Buffer, and centrifuging at 10000rpm for 1 min;
(5) repeating the step 4 once, and centrifuging at 12000rpm for 2 min;
(6) add 50ul of 65 ℃ preheated Elution Buffer, stand for 2min, centrifuge at 12000rpm for 2 min.
1.6 ligation and transformation
The enzyme-digested HCVNS3 gene and pMAl-C are mixed2The skeleton carrier is connected, and the connection system is as follows:
and (3) transforming escherichia coli: adding the ligation product into the competence of Escherichia coli DH5a, mixing uniformly, and carrying out ice bath for 30 min. ② the water bath heat shock at 42 ℃ is converted for 90s, and the mixture is immediately put back on ice and is placed for 3 min. ③ 600ul of LB medium was added into the centrifuge tube, and the mixture was cultured for 1 hour at 37C170rpm with shaking.
And fourthly, centrifuging at 3000rpm for 10min, sucking the supernatant and only keeping about 100ul, uniformly blowing, spreading on a solid LB plate containing Amp resistance, placing in an incubator at 37 ℃ for 30min in an upright mode, and culturing in an inverted mode overnight after the bacterial liquid is absorbed.
And (4) selecting monoclonal culture and plasmid extraction, and sending the extracted plasmid to a company for sequencing, wherein the result is correct.
1.7 prokaryotic expression
Taking 50ul of escherichia coli BL21 competence, carrying out ice bath, adding 2ul of plasmid, and carrying out ice bath for 30 min;
② heat shocking at 42 ℃ for 90s, then ice-cooling for 3min, adding 600ul LB liquid culture medium, shaking and culturing at 37 ℃ and 180rpm for 1 h.
③ centrifuging at 10000rpm for 1min, sucking the supernatant, reserving about 100ul, mixing uniformly and coating on Amp resistant LB plate, placing in a 37 ℃ incubator for 30min, and culturing overnight in an inverted way.
1.8 expression and cell treatment
Colonies were picked from the Amp-resistant plate of 1.7, transferred to an Amp-resistant liquid medium, and cultured overnight at 37 ℃ with shaking at 180 rpm.
② weighing the overnight cultured bacterial liquidNewly transferred into Amp resistant liquid culture medium, and subjected to shaking culture at 37 deg.C and 220rpm for 4h at OD600When the temperature was decreased to 16 ℃ and the cells were cultured at 180rpm for 1 hour with shaking, IPTG (1 mM final concentration) was added thereto, and the cells were cultured overnight.
Thirdly, centrifuging at 8000rpm for 10min, collecting thalli, adding 50ml PBS/L for resuspension, adjusting the pressure of a high-pressure crusher to 800bar, crushing for 3 times, centrifuging at 12000rpm for 40min, and taking supernatant to obtain supernatant after thalli are crushed.
1.9 MBP column purification
(1) Dripping out the preservation solution in the MBP column, adding 10ml of water to clean the MBP column, and repeating for 2 times;
(2) adding 10ml of MBP equilibrium solution to balance the column, and repeating twice;
(3) adding the supernatant obtained after the 1.8 crushed thalli into an MBP column;
(4) washing the impure protein twice by using 10ml of balance liquid to fill the column;
(5) eluting protein with 4ml of eluent, and collecting.
1.10 SDS-PAGE detection of proteins
24ul of purified protein (i.e., HCV Core/NS3 fusion protein) was placed in a 1.5ml centrifuge tube, 6ul of 5 × SDS loading buffer was added proportionally, mixed well and boiled in boiling water for 10 min. 10ul of the treated sample was loaded. The electrophoresis device is connected with a power supply for 120V and 120 min.
The gel was removed from the electrophoresis apparatus, placed in a coomassie brilliant blue staining solution, heated in a microwave oven for 3min, transferred to a destaining solution, heated in a microwave oven until the background was clear, and the results were observed and recorded, see fig. 2.
1.11 Secondary purification of proteins
Purification was carried out using the kit Dextrin Sepharose High Performance (Lot:10288766) from GE according to the instructions. Thus obtaining the purified HCV Core/NS3 fusion protein.
Example 2 preparation of colloidal gold test strip
A colloidal gold test strip, as shown in figure 5, a sample loading pad 2, a colloidal gold adsorption pad 3, a carrier film 4 and a water absorption pad 5 are overlapped and lapped on a substrate 1 in sequence;
the colloidal gold adsorption pad 3 is coated with colloidal gold-labeled HCV Core/NS3 fusion protein and colloidal gold-labeled rabbit IgG.
The bearing film 4 is provided with a detection line T and a quality control line C which are spaced, the detection line is arranged at one side close to the colloidal gold adsorption pad 3, and the quality control line C is arranged at one side close to the water absorption pad 5; the detection line T is coated with HCV Core/NS3 fusion protein, and the quality control line C is coated with goat rabbit IgG. The distance between the detection line T and the quality control line C is 4-6mm, and the distance is selected to be 4mm in the embodiment.
Further, the sample loading pad 2 and the absorbent pad 5 are both provided with a protective film 6.
The preparation method of the colloidal gold test strip comprises the following steps:
1. preparing a carrier film:
(1) selecting a nitrocellulose membrane with the aperture of 3-10 mu m, and cutting the membrane into specifications with the width of 2.0cm and the length of 30.5cm according to requirements for later use.
(2) Preparing fusion protein for coating the detection line by using 0.1M, pH 8.0.0 Tris-HCL buffer solution, wherein the concentration of the fusion protein is 0.3-2mg/ml, and in the embodiment, the concentration is 1 mg/ml; sodium chloride buffer solution with the mass percentage content of 0.85% is used for preparing goat anti-rabbit IgG for quality control line coating, so that the antibody concentration is 0.5-3mg/ml, and in the embodiment, the antibody concentration is 2 mg/ml.
(3) Selecting an antibody coating surface of a nitrocellulose membrane and marking, uniformly coating an antibody (antigen) solution of a detection line and a quality control line which need to be coated on a membrane in parallel, wherein the distance between the detection line and the quality control line is as follows: 4.0mm, and drying the nitrocellulose membrane at the constant temperature of 2-30 ℃ for later use.
(4) Preparing a sealing treatment soaking solution, and adding purified water with actual production capacity into a liquid preparation tank; respectively weighing buffer solution, sugar, blocking protein and preservative, directly adding the above components into a liquid preparation tank, stirring until the components are completely dissolved, adding purified water to a desired volume, and fully and uniformly stirring for at least 10 minutes for later use; the prepared sealing treatment soaking solution contains 0.1Mol of buffer solution, 0.5 mass percent of sugar, 1 mass percent of sealing protein and 0.05 mass percent of preservative, wherein the buffer solution is phosphate buffer solution, the sugar is cane sugar, the preservative is thimerosal, and the sealing protein is bovine serum albumin.
(5) Placing the membranes coated with the detection lines and the quality control lines in a treatment tank, adding the prepared sealing treatment soaking liquid of the step (4), ensuring that each membrane is completely immersed in the sealing treatment soaking liquid of the step (4) for 30 minutes, ensuring that the membranes do not move and overlap, taking out the membranes from the treatment tank after 30 minutes, and pouring the sealing treatment soaking liquid of the step (4); and (4) placing the membrane on gauze by using tweezers, and airing to be slightly dry to obtain the carrier membrane.
(6) Pasting a board and drying: tearing off white paper in the middle of a cutting line on double-sided adhesive of a rubber plate, putting a closed bearing film right at the central blank position of the rubber plate by using tweezers, enabling the right side of the rubber plate to be flush with the right side of the film, avoiding errors in the production process, ensuring that the color development position is relatively accurate, pasting one end of all top quality control lines when pasting the rubber plate, smearing the film surface through the double-sided adhesive paper after pasting the film on the rubber plate, avoiding bubbles, controlling the indoor temperature to be 18-28 ℃ and the relative humidity to be less than or equal to 40%, and ensuring that air can circulate in a drying room and air of a dehumidifier can not directly blow on the film surface. The drying time is more than or equal to 4 hours and is reserved.
2. Preparation of colloidal gold adsorption pad
(1) Preparing a colloidal gold complex solution: adding an actual production amount of purified water to the liquor preparation tank; trehalose, bovine serum albumin, trisodium citrate, polyethylene glycol and NaN were weighed using an electronic analytical balance3Directly adding the colloidal gold complex solution into a liquid preparation tank for stirring until the colloidal gold complex solution is completely dissolved, adding purified water to a constant volume to a required volume, fully and uniformly stirring, wherein the stirring time is more than 30 minutes, and the obtained colloidal gold complex solution contains 5 mass percent of trehalose, 2 mass percent of bovine serum albumin, 0.5 mass percent of trisodium citrate, 0.05 mass percent of polyethylene glycol and 0.05 mass percent of NaN3。
(2) Measuring the colloidal gold with the required labeling amount by using a measuring cylinder, adjusting according to the pH value of 7.0-7.3, adding 0.2mol/L potassium carbonate solution according to the volume percentage content of 0.50 percent, stirring for 15 minutes on a magnetic stirrer, taking the fusion protein, diluting the fusion protein by double distilled water, and labeling colloidal gold at 10ug/ml (i.e., adding colloidal gold to the fusion protein diluent to give a final concentration of 10ug/ml), adding the fusion protein to the colloidal gold, stirring for 30 minutes on a magnetic stirrer, adding stabilizer polyethylene glycol with the volume percentage of 0.5 per mill, stirring for 30 minutes, centrifuging, collecting precipitate, redissolving by using the colloidal gold composite solution with the volume percentage of 3 percent, stirring on the magnetic stirrer until the mixture is uniformly mixed to obtain the colloidal gold-fusion protein conjugate composite solution with the volume percentage of 3 percent for later use.
(3) Measuring the colloidal gold with the required labeling amount by using a measuring cylinder, adjusting according to the pH value of 7.0-7.3, namely adding 0.2mol/L potassium carbonate solution according to the volume percentage of 0.50%, stirring for 15 minutes on a magnetic stirrer, taking rabbit IgG, diluting the rabbit IgG by using double distilled water, labeling the colloidal gold according to 20ug/ml, adding the rabbit IgG into the colloidal gold, stirring for 30 minutes on the magnetic stirrer, adding 0.5 thousandth of stabilizer polyethylene glycol according to the volume percentage, stirring for 30 minutes, centrifuging, collecting precipitate, redissolving by using 3% of colloidal gold redissolution according to the volume percentage, stirring on the magnetic stirrer until the mixture is uniform, and obtaining the 3% colloidal gold-rabbit IgG conjugate redissolution for later use.
(4) Taking the colloidal gold-fusion protein conjugate compound solution with the volume percentage of 3% and the colloidal gold-rabbit IgG antibody conjugate compound solution with the volume percentage of 50% in the above (2) and (3), re-dissolving the colloidal gold re-solution with the volume percentage of 50%, uniformly mixing the re-dissolved colloidal gold-fusion protein conjugate compound solution and the colloidal gold-rabbit IgG antibody conjugate compound solution on a magnetic stirrer according to the volume percentage of 50 mu L/cm2Pouring the colloidal gold onto a prepared colloidal gold adsorption pad, placing the colloidal gold adsorption pad in a drying chamber to be dried for more than or equal to 4 hours, controlling the temperature of the drying chamber to be 18-28 ℃, controlling the relative humidity to be less than or equal to 40%, ensuring smooth air and preventing airflow from directly blowing on the colloidal gold adsorption pad, placing the dried colloidal gold adsorption pad into an aluminum foil bag filled with a drying agent, sealing and storing, and making a labeled colloidal gold adsorption pad for later use.
Note: the colloidal gold is cast because the cast colloidal gold can be freely cut in width, thereby being convenient for adjusting the color development of the product.
3. Assembling and cutting:
(1) taking a semi-finished product of the transparent substrate adhered with the bearing film, cutting the test strip colloidal gold adsorption pad into strips of 0.5cm multiplied by 30cm according to the width of 0.5cm, adhering the strips on the transparent substrate close to one side of the detection line, keeping the strips in lap joint with the bearing film for about 1mm, compounding the test strip water absorption pad on the transparent substrate close to one side of the quality control line and in lap joint with the bearing film for about 1mm, compounding the test strip sample loading pad on one end, far away from the bearing film, of the test strip colloidal gold adsorption pad and in lap joint with the end for about 1mm, and marking for later use;
(2) and cutting the assembled substrate into strip test paper for later use.
Example 3 detection of hepatitis C Using colloidal gold test strip
1000 parts of negative specimen (from the disease prevention and control center in Henan province, the third 0 th hospital of the people's liberation force in China and the third 0 th hospital of the people's society in Subei) was taken, and the result of detection by the colloidal gold test paper prepared in example 2 showed that the negative coincidence rate was more than 95%, and FIG. 3 shows the detection result of a part of negative specimen.
100 parts of positive specimens (from the disease prevention and control center in Henan province, the third 0 th Hospital of the people's liberation force in China and the Subei Hospital) were collected and tested using the colloidal gold test paper prepared in example 2. The positive detection rate is more than 96%, and fig. 4 shows the detection result of part of positive specimens.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Sequence listing
<110> Nantong Yishi Biotechnology Ltd
<120> fusion protein and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 555
<212> PRT
<213> Artificial sequence
<400> 1
Met Ser Thr Ala Pro Leu Pro Gly Ala Leu Thr Leu Ala Ala Thr Ala
1 5 10 15
Ala Ala Pro Gly Ala Val Leu Pro Pro Gly Gly Gly Gly Ile Val Gly
20 25 30
Gly Val Thr Leu Leu Pro Ala Ala Gly Pro Ala Leu Gly Val Ala Ala
35 40 45
Thr Ala Leu Thr Ser Gly Ala Ser Gly Pro Ala Gly Ala Ala Gly Pro
50 55 60
Ile Pro Leu Ala Ala Ala Pro Gly Gly Ala Thr Thr Ala Gly Pro Gly
65 70 75 80
Thr Pro Thr Pro Leu Thr Gly Ala Gly Gly Met Gly Thr Ala Gly Thr
85 90 95
Leu Leu Ser Pro Ala Gly Ser Ala Pro Ser Thr Gly Pro Thr Ala Pro
100 105 110
Ala Ala Ala Ser Ala Ala Leu Gly Leu Val Ile Ala Thr Leu Thr Cys
115 120 125
Gly Pro Ala Ala Leu Met Gly Thr Ile Pro Leu Val Gly Ala Pro Leu
130 135 140
Gly Gly Ala Ala Ala Ala Leu Ala His Gly Val Ala Val Leu Gly Ala
145 150 155 160
Gly Ala His Thr Ala Ser Ser Ser Val Pro Gly Ala Gly Gly Gly Gly
165 170 175
Ser Gly Gly Gly Gly Ser Leu Gly Ile Gly Thr Val Leu Ala Gly Ala
180 185 190
Gly Thr Ala Gly Val Ala Leu Val Val Leu Ala Val Thr Val Pro His
195 200 205
His Ala Ile Gly Gly Val Ala Leu Thr Ala Ala Gly Gly Ile Pro Pro
210 215 220
Thr Gly Leu Ala Ile Pro Leu Gly Thr Ile Leu Gly Gly Ala His Leu
225 230 235 240
Ile Pro Cys His Ser Leu Leu Leu Cys Ala Gly Leu Ala Ala Ala Leu
245 250 255
Ser Ala Leu Gly Leu Ala Ala Val Ala Thr Thr Ala Gly Leu Ala Val
260 265 270
Ser Val Ile Pro Thr Ser Gly Ala Val Val Val Val Ala Thr Ala Ala
275 280 285
Leu Met Thr Gly Thr Thr Gly Ala Pro Ala Ser Val Ile Ala Cys Ala
290 295 300
Thr Cys Val Thr Gly Thr Val Ala Pro Ser Leu Ala Pro Thr Pro Thr
305 310 315 320
Ile Gly Thr Thr Thr Met Pro Gly Ala Ala Val Ser Ala Ser Gly Ala
325 330 335
Ala Gly Ala Thr Gly Ala Gly Ala Gly Gly Ile Thr Ala Pro Val Thr
340 345 350
Pro Gly Gly Ala Pro Ser Gly Met Pro Ala Ser Ser Val Leu Cys Gly
355 360 365
Cys Thr Ala Ala Gly Cys Ala Thr Thr Gly Leu Thr Pro Ala Gly Thr
370 375 380
Ser Val Ala Leu Ala Ala Thr Leu Ala Thr Pro Gly Leu Pro Val Cys
385 390 395 400
Gly Ala His Leu Gly Pro Thr Gly Ser Val Pro Thr Gly Leu Thr His
405 410 415
Ile Ala Ala His Pro Leu Ser Gly Thr Leu Gly Ala Gly Ala Ala Pro
420 425 430
Pro Thr Leu Val Ala Thr Gly Ala Thr Val Cys Ala Ala Ala Gly Thr
435 440 445
Ala Gly Met Thr Leu Cys Leu Ile Ala Leu Leu Pro Thr Leu His Gly
450 455 460
Pro Thr Pro Leu Leu Thr Ala Leu Gly Ala Val Gly Ala Gly Val Thr
465 470 475 480
Leu Thr His Pro Leu Thr Leu Thr Ile Met Ala Cys Met Ser Ala Ala
485 490 495
Leu Gly Val Val Thr Ser Thr Thr Val Leu Val Gly Gly Val Leu Ala
500 505 510
Ala Leu Ala Ala Thr Cys Leu Thr Thr Gly Ser Val Val Ile Val Gly
515 520 525
Ala Ile Ile Leu Ser Gly Leu Pro Ala Val Ile Pro Ala Ala Ala Val
530 535 540
Leu Thr Ala Gly Pro Ala Gly Met Gly Gly Cys
545 550 555
<210> 2
<211> 1668
<212> DNA
<213> Artificial sequence
<400> 2
atgagcacca atccgaaacc gcagcgcaaa accaaacgta ataccaatcg ccgcccgcag 60
gatgtgaaat ttccgggtgg tggccagatt gtgggtggtg tttatctgct gccgcgtcgt 120
ggcccgcgtc tgggtgtgcg tgcaacacgt aaaaccagtg aacgtagcca gccgcgtggc 180
cgtcgtcagc ctattccgaa agcacgtcgt ccggaaggcc gcacctgggc acaacctggt 240
tatccgtggc cgctgtatgg taatgaaggt atgggttggg caggttggct gctgagtccg 300
cgtggcagcc gtcctagctg gggtcctacc gatccgcgtc gtcgcagccg taatctgggt 360
aaagtgattg ataccctgac ctgtggcttt gccgatctga tgggctatat tccgctggtt 420
ggcgccccgc tgggcggtgc agcacgtgca ttagcccacg gtgttcgcgt gctggaagat 480
ggcgcacatt ggaatagcag cagtgtgccg ggcgatggcg gcggtggtag tggtggtggt 540
ggctcactgg gtattggtac agtgctggat caggcagaaa ccgcaggcgt tcgtctggtg 600
gttctggcag tgaccgtgcc gcatcataat attgaagaag ttgccctgac caatgatggt 660
gaaattccgt tttatggtaa ggcaattccg ctggaaacca ttaaaggcgg tcgccatctg 720
attttttgtc atagtaaaaa gaagtgcgac gagctggcag cacgcctgag cgctctgggt 780
ctgaatgcag ttgcctatta tcgtggcctg gatgttagcg tgattccgac cagcggtgat 840
gtggtggtgg ttgccaccga tgcactgatg accggctata ccggcgattt tgatagcgtg 900
attgattgta atacctgcgt gacccagacc gtggatttta gtctggaccc tacctttacc 960
attgaaacca ccaccatgcc gcaggatgcc gtgagccgca gccagagacg cggtagaacc 1020
ggccgtggtc gtggcggtat ttatcgtttt gttacccctg gtgaacgtcc gagtggcatg 1080
tttgatagta gtgttctgtg tgaatgctac gatgcaggtt gtgcctggta tgaactgacc 1140
ccggcagaaa ccagcgtgcg tctgcgtgca tatctgaata cccctggtct gccggtttgt 1200
caggatcatc tggaattttg ggaaagtgtg tttaccggcc tgacccatat tgatgcccat 1260
tttctgagtc agaccaaaca ggccggcgat aattttccgt atctggttgc atatcaggcc 1320
accgtttgtg cacgtgcaca gtgggatcag atgtggaaat gcctgattcg tctgaaaccg 1380
accctgcatg gtccgacccc gctgttatat cgtctgggcg cagtgcagaa tgaagtgacc 1440
ctgacccatc cgaaaaccaa atatattatg gcatgcatga gcgccgatct ggaagtggtg 1500
accagcacct gggtgctggt tggcggtgtg ctggcagcac tggccgcata ttgcctgacc 1560
accggtagcg ttgtgattgt gggtcgtatt attctgagcg gcaaaccggc agttattccg 1620
gatcgtgatg ttctgtatcg tgaatttgat gaaatggagg aatgctaa 1668
<210> 3
<211> 27
<212> DNA
<213> Artificial sequence
<400> 3
ccggaattca tgagcaccaa tccgaaa 27
<210> 4
<211> 30
<212> DNA
<213> Artificial sequence
<400> 4
cccaagcttt tagcattcct ccatttcatc 30
Claims (5)
1. Any of the following products:
(B1) a fusion protein is composed of hepatitis C virus core protein and hepatitis C virus non-structural protein NS 3;
(B2) a nucleic acid molecule encoding the fusion protein;
(B3) an expression cassette comprising the nucleic acid molecule of (B2);
(B4) a recombinant vector comprising (B2) the nucleic acid molecule or a recombinant vector comprising (B3) the expression cassette;
(B5) a recombinant microorganism containing (B2) the nucleic acid molecule, or a recombinant microorganism containing (B3) the expression cassette, or a recombinant microorganism containing B4) the recombinant vector.
2. The product of claim 1, wherein the hepatitis c virus core protein and the hepatitis c virus nonstructural protein NS3 are linked by a linker peptide.
3. The product of claim 1 or 2, wherein the amino acid sequence of the fusion protein consists of the amino acid sequence of the hepatitis C virus core protein, the amino acid sequence of the linker peptide, and the amino acid sequence of the hepatitis C virus non-structural protein NS3 in that order from N-terminus to C-terminus;
or the like, or, alternatively,
the amino acid sequence of the fusion protein consists of an amino acid sequence of a tag protein, methionine, an amino acid sequence of a core protein of the hepatitis C virus, an amino acid sequence of a connecting peptide and an amino acid sequence of a non-structural protein NS3 of the hepatitis C virus in sequence from the N terminal to the C terminal.
4. The product of any one of claims 1-3, wherein the hepatitis C virus core protein amino acid sequence is SEQ ID No.2, positions 2-172; the nucleotide sequence of the coding DNA molecule is SEQ ID No.1(5 '-3') 4-516;
the amino acid sequence of the hepatitis C virus non-structural protein NS3 is 182-555 bit of SEQ ID No. 2; the nucleotide sequence of the coding DNA molecule is SEQ ID No.1(5 '-3') at position 544-1665;
the connecting peptide has the amino acid sequence of 173-181 of SEQ ID No. 2; the nucleotide sequence of the coding DNA molecule is 517-543 th site of SEQ ID No.1(5 '-3');
the tag protein is an MBP tag.
5. The fusion protein is applied to detecting hepatitis C.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110984130.3A CN113667022B (en) | 2021-08-25 | 2021-08-25 | Fusion protein and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110984130.3A CN113667022B (en) | 2021-08-25 | 2021-08-25 | Fusion protein and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113667022A true CN113667022A (en) | 2021-11-19 |
CN113667022B CN113667022B (en) | 2024-03-22 |
Family
ID=78546329
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110984130.3A Active CN113667022B (en) | 2021-08-25 | 2021-08-25 | Fusion protein and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113667022B (en) |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050053915A1 (en) * | 1999-08-27 | 2005-03-10 | Penelope Mavromara | Nucleic acids and new polypeptides associated with and/or overlapping with hepatitis C virus core gene products |
CN102262158A (en) * | 2010-05-27 | 2011-11-30 | 戴国祯 | Direct application of recombinant fusion protein to quick diagnostic detection |
CN102305862A (en) * | 2011-05-25 | 2012-01-04 | 方辉 | Method for detecting hepatitis C virus antibody in serum |
CN102321179A (en) * | 2011-08-10 | 2012-01-18 | 杭州培乐生物技术有限公司 | Hepatitis C virus recombination protein and gene sequence |
CN104697988A (en) * | 2015-02-10 | 2015-06-10 | 深圳市新产业生物医学工程股份有限公司 | Kit for detecting hepatitis c virus antibody as well as detection method and application thereof |
CN108530521A (en) * | 2018-04-18 | 2018-09-14 | 南京京达生物技术有限公司 | Recombinate the preparation and application of hepatitis C antigen |
-
2021
- 2021-08-25 CN CN202110984130.3A patent/CN113667022B/en active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050053915A1 (en) * | 1999-08-27 | 2005-03-10 | Penelope Mavromara | Nucleic acids and new polypeptides associated with and/or overlapping with hepatitis C virus core gene products |
CN102262158A (en) * | 2010-05-27 | 2011-11-30 | 戴国祯 | Direct application of recombinant fusion protein to quick diagnostic detection |
CN102305862A (en) * | 2011-05-25 | 2012-01-04 | 方辉 | Method for detecting hepatitis C virus antibody in serum |
CN102321179A (en) * | 2011-08-10 | 2012-01-18 | 杭州培乐生物技术有限公司 | Hepatitis C virus recombination protein and gene sequence |
CN104697988A (en) * | 2015-02-10 | 2015-06-10 | 深圳市新产业生物医学工程股份有限公司 | Kit for detecting hepatitis c virus antibody as well as detection method and application thereof |
CN108530521A (en) * | 2018-04-18 | 2018-09-14 | 南京京达生物技术有限公司 | Recombinate the preparation and application of hepatitis C antigen |
Also Published As
Publication number | Publication date |
---|---|
CN113667022B (en) | 2024-03-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112079906B (en) | Novel coronavirus spike protein, related biological material, application of novel coronavirus spike protein, detection test paper and detection kit | |
CN1609617B (en) | Composition and method for diagnosing and preventing serious acute respiratory syndrome(SARS) | |
CN103760365B (en) | A kind of BAS-ELISA kit detecting CSFV Erns and E2 antigen | |
CN104826100A (en) | Preparation method and application of classical swine fever virus recombinant subunit vaccine | |
CN106543272A (en) | Multifunctional label fusion protein and its expression and application | |
EP0480041A1 (en) | Fused antigen polypeptide | |
CN116199786A (en) | Fusion protein and application thereof in helicobacter pylori CagA antibody detection | |
CN113667022A (en) | Fusion protein and application thereof | |
CN113640519B (en) | Kit for specifically detecting hepatitis C and preparation method thereof | |
CN106955361A (en) | A kind of pharmaceutical composition containing tuberculosis allergen CE | |
KR102100813B1 (en) | Fusion protein from hepatitis C virus and diagnosing kit comprising the same | |
CN112724247A (en) | SARS-CoV-2 coronavirus IgA antibody and its application in ELISA detection kit | |
CN110927374A (en) | Colloidal gold test strip for detecting hepatitis E virus IgG antibody and preparation method thereof | |
CN106754981A (en) | A kind of method that utilization E. coli system prepares Goose Parvovirus sample particle | |
CN116203233A (en) | Helicobacter pylori CagA antibody detection test paper and application thereof | |
CN115166235A (en) | African swine fever virus double-antigen sandwich antibody detection kit and preparation method thereof | |
CN106636132B (en) | A kind of ultraviolet stress-related genes of highland barley and its application | |
CN106167523A (en) | A kind of Irisin recombiant protein and synthetic method thereof | |
CN106397546A (en) | O type foot-and-mouth disease virus artificial recombinant antigen and preparation and application thereof | |
CN117924439B (en) | Recombinant CagA protein and preparation method and application thereof | |
CN102993283B (en) | Antigen protein for mycobacterium tuberculosis and application | |
CN105601749A (en) | Recombinant CssPLA2 protein and soluble expression in vitro and purifying preparation method thereof | |
CN108359680A (en) | Recombinant plasmid, genetic engineering bacterium containing human cytomegalovirus UL146 genes and its application | |
CN113881690B (en) | Fusion plasmid containing gene for encoding Tachenopodium album 2 nucleoprotein, host bacteria and preparation method and application of protein | |
CN110540602A (en) | Toxoplasma gondii surface antigen GRA1 and GRA7 recombinant protein colloidal gold test strip |
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 |