CN110577593B - 一种小分子近红外光荧光蛋白及其融合蛋白 - Google Patents
一种小分子近红外光荧光蛋白及其融合蛋白 Download PDFInfo
- Publication number
- CN110577593B CN110577593B CN201910586801.3A CN201910586801A CN110577593B CN 110577593 B CN110577593 B CN 110577593B CN 201910586801 A CN201910586801 A CN 201910586801A CN 110577593 B CN110577593 B CN 110577593B
- Authority
- CN
- China
- Prior art keywords
- ala
- val
- leu
- gly
- glu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108091006047 fluorescent proteins Proteins 0.000 title claims abstract description 83
- 102000034287 fluorescent proteins Human genes 0.000 title claims abstract description 83
- 150000003384 small molecules Chemical class 0.000 title abstract description 6
- 108020001507 fusion proteins Proteins 0.000 title description 5
- 102000037865 fusion proteins Human genes 0.000 title description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 12
- 230000004927 fusion Effects 0.000 claims description 16
- 238000003384 imaging method Methods 0.000 claims description 11
- 102000039446 nucleic acids Human genes 0.000 claims description 9
- 108020004707 nucleic acids Proteins 0.000 claims description 9
- 150000007523 nucleic acids Chemical class 0.000 claims description 9
- 239000013598 vector Substances 0.000 claims description 7
- 241001465754 Metazoa Species 0.000 claims description 6
- 230000004807 localization Effects 0.000 claims description 3
- 230000001413 cellular effect Effects 0.000 claims 1
- 230000035772 mutation Effects 0.000 abstract description 12
- 150000001413 amino acids Chemical group 0.000 abstract description 9
- 239000000178 monomer Substances 0.000 abstract description 9
- 230000012743 protein tagging Effects 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 description 30
- 108010073969 valyllysine Proteins 0.000 description 29
- 102000004169 proteins and genes Human genes 0.000 description 28
- 235000018102 proteins Nutrition 0.000 description 26
- GWZYPXHJIZCRAJ-UHFFFAOYSA-N Biliverdin Natural products CC1=C(C=C)C(=C/C2=NC(=Cc3[nH]c(C=C/4NC(=O)C(=C4C)C=C)c(C)c3CCC(=O)O)C(=C2C)CCC(=O)O)NC1=O GWZYPXHJIZCRAJ-UHFFFAOYSA-N 0.000 description 22
- RCNSAJSGRJSBKK-NSQVQWHSSA-N Biliverdin IX Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(\C=C/2C(=C(C)C(=C/C=3C(=C(C=C)C(=O)N=3)C)/N\2)CCC(O)=O)N1 RCNSAJSGRJSBKK-NSQVQWHSSA-N 0.000 description 22
- QBUVFDKTZJNUPP-UHFFFAOYSA-N biliverdin-IXalpha Natural products N1C(=O)C(C)=C(C=C)C1=CC1=C(C)C(CCC(O)=O)=C(C=C2C(=C(C)C(C=C3C(=C(C=C)C(=O)N3)C)=N2)CCC(O)=O)N1 QBUVFDKTZJNUPP-UHFFFAOYSA-N 0.000 description 22
- 210000004027 cell Anatomy 0.000 description 22
- YSUVMPICYVWRBX-VEVYYDQMSA-N Arg-Asp-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O YSUVMPICYVWRBX-VEVYYDQMSA-N 0.000 description 15
- PSOPJDUQUVFSLS-GUBZILKMSA-N Arg-Met-Cys Chemical compound CSCC[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N PSOPJDUQUVFSLS-GUBZILKMSA-N 0.000 description 15
- VMVUDJUXJKDGNR-FXQIFTODSA-N Asp-Met-Asn Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)O)N VMVUDJUXJKDGNR-FXQIFTODSA-N 0.000 description 15
- OTKUAVXGMREHRX-CFMVVWHZSA-N Asp-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 OTKUAVXGMREHRX-CFMVVWHZSA-N 0.000 description 15
- MHYHLWUGWUBUHF-GUBZILKMSA-N Cys-Val-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CS)N MHYHLWUGWUBUHF-GUBZILKMSA-N 0.000 description 15
- LPHGXOWFAXFCPX-KKUMJFAQSA-N Glu-Pro-Phe Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CCC(=O)O)N)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)O LPHGXOWFAXFCPX-KKUMJFAQSA-N 0.000 description 15
- QCMVGXDELYMZET-GLLZPBPUSA-N Glu-Thr-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O QCMVGXDELYMZET-GLLZPBPUSA-N 0.000 description 15
- VIIBEIQMLJEUJG-LAEOZQHASA-N Gly-Ile-Gln Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(O)=O VIIBEIQMLJEUJG-LAEOZQHASA-N 0.000 description 15
- UWQDKRIZSROAKS-FJXKBIBVSA-N Gly-Met-Thr Chemical compound [H]NCC(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UWQDKRIZSROAKS-FJXKBIBVSA-N 0.000 description 15
- CCQLQKZTXZBXTN-NHCYSSNCSA-N Leu-Gly-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O CCQLQKZTXZBXTN-NHCYSSNCSA-N 0.000 description 15
- IBSGMIPRBMPMHE-IHRRRGAJSA-N Leu-Met-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(O)=O IBSGMIPRBMPMHE-IHRRRGAJSA-N 0.000 description 15
- IMAKMJCBYCSMHM-AVGNSLFASA-N Lys-Glu-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN IMAKMJCBYCSMHM-AVGNSLFASA-N 0.000 description 15
- WAIHHELKYSFIQN-XUXIUFHCSA-N Lys-Ile-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O WAIHHELKYSFIQN-XUXIUFHCSA-N 0.000 description 15
- 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 15
- JSGWNFKWZNPDAV-YDHLFZDLSA-N Phe-Val-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 JSGWNFKWZNPDAV-YDHLFZDLSA-N 0.000 description 15
- SCZJKZLFSSPJDP-ACRUOGEOSA-N Tyr-Phe-Leu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O SCZJKZLFSSPJDP-ACRUOGEOSA-N 0.000 description 15
- ASQFIHTXXMFENG-XPUUQOCRSA-N Val-Ala-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O ASQFIHTXXMFENG-XPUUQOCRSA-N 0.000 description 15
- OVBMCNDKCWAXMZ-NAKRPEOUSA-N Val-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](C(C)C)N OVBMCNDKCWAXMZ-NAKRPEOUSA-N 0.000 description 15
- 108010038633 aspartylglutamate Proteins 0.000 description 15
- 230000015572 biosynthetic process Effects 0.000 description 15
- 108010054155 lysyllysine Proteins 0.000 description 15
- 238000003786 synthesis reaction Methods 0.000 description 15
- NKLRWRRVYGQNIH-GHCJXIJMSA-N Asn-Ile-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O NKLRWRRVYGQNIH-GHCJXIJMSA-N 0.000 description 14
- KKZHXOOZHFABQQ-UWJYBYFXSA-N Cys-Ala-Tyr Chemical compound SC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 KKZHXOOZHFABQQ-UWJYBYFXSA-N 0.000 description 14
- KXRORHJIRAOQPG-SOUVJXGZSA-N Glu-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CCC(=O)O)N)C(=O)O KXRORHJIRAOQPG-SOUVJXGZSA-N 0.000 description 14
- SOYWRINXUSUWEQ-DLOVCJGASA-N Glu-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCC(O)=O SOYWRINXUSUWEQ-DLOVCJGASA-N 0.000 description 14
- LJPIRKICOISLKN-WHFBIAKZSA-N Gly-Ala-Ser Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O LJPIRKICOISLKN-WHFBIAKZSA-N 0.000 description 14
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 14
- 241000880493 Leptailurus serval Species 0.000 description 14
- XBBKIIGCUMBKCO-JXUBOQSCSA-N Leu-Ala-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XBBKIIGCUMBKCO-JXUBOQSCSA-N 0.000 description 14
- ULXYQAJWJGLCNR-YUMQZZPRSA-N Leu-Asp-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O ULXYQAJWJGLCNR-YUMQZZPRSA-N 0.000 description 14
- KQFZKDITNUEVFJ-JYJNAYRXSA-N Leu-Phe-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)CC1=CC=CC=C1 KQFZKDITNUEVFJ-JYJNAYRXSA-N 0.000 description 14
- VKVDRTGWLVZJOM-DCAQKATOSA-N Leu-Val-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O VKVDRTGWLVZJOM-DCAQKATOSA-N 0.000 description 14
- PLOUVAYOMTYJRG-JXUBOQSCSA-N Lys-Thr-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O PLOUVAYOMTYJRG-JXUBOQSCSA-N 0.000 description 14
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 14
- HAAQQNHQZBOWFO-LURJTMIESA-N Pro-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H]1CCCN1 HAAQQNHQZBOWFO-LURJTMIESA-N 0.000 description 14
- FXGIMYRVJJEIIM-UWVGGRQHSA-N Pro-Leu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 FXGIMYRVJJEIIM-UWVGGRQHSA-N 0.000 description 14
- PIQRHJQWEPWFJG-UWJYBYFXSA-N Ser-Tyr-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O PIQRHJQWEPWFJG-UWJYBYFXSA-N 0.000 description 14
- GBIUHAYJGWVNLN-UHFFFAOYSA-N Val-Ser-Pro Natural products CC(C)C(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O GBIUHAYJGWVNLN-UHFFFAOYSA-N 0.000 description 14
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 14
- 108010011559 alanylphenylalanine Proteins 0.000 description 14
- 108010084264 glycyl-glycyl-cysteine Proteins 0.000 description 14
- 108700042769 prolyl-leucyl-glycine Proteins 0.000 description 14
- PCIFXPRIFWKWLK-YUMQZZPRSA-N Ala-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N PCIFXPRIFWKWLK-YUMQZZPRSA-N 0.000 description 13
- OISWSORSLQOGFV-AVGNSLFASA-N Arg-Met-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@@H](N)CCCN=C(N)N OISWSORSLQOGFV-AVGNSLFASA-N 0.000 description 13
- BWMMKQPATDUYKB-IHRRRGAJSA-N Arg-Tyr-Asn Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(O)=O)CC1=CC=C(O)C=C1 BWMMKQPATDUYKB-IHRRRGAJSA-N 0.000 description 13
- XDKKMRPRRCOELJ-GUBZILKMSA-N Pro-Val-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 XDKKMRPRRCOELJ-GUBZILKMSA-N 0.000 description 13
- QYDKSNXSBXZPFK-ZJDVBMNYSA-N Thr-Thr-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QYDKSNXSBXZPFK-ZJDVBMNYSA-N 0.000 description 13
- VHIZXDZMTDVFGX-DCAQKATOSA-N Val-Ser-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N VHIZXDZMTDVFGX-DCAQKATOSA-N 0.000 description 13
- 235000001014 amino acid Nutrition 0.000 description 13
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 12
- 239000004475 Arginine Substances 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 125000000539 amino acid group Chemical group 0.000 description 8
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 8
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 229960000789 guanidine hydrochloride Drugs 0.000 description 6
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 6
- 238000001228 spectrum Methods 0.000 description 6
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical group CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 239000004474 valine Chemical group 0.000 description 5
- 238000001042 affinity chromatography Methods 0.000 description 4
- 230000002776 aggregation Effects 0.000 description 4
- 238000004220 aggregation Methods 0.000 description 4
- 238000005286 illumination Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 108060006184 phycobiliprotein Proteins 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- 102000006410 Apoproteins Human genes 0.000 description 3
- 108010083590 Apoproteins Proteins 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 3
- 210000003483 chromatin Anatomy 0.000 description 3
- 238000002270 exclusion chromatography Methods 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000003595 spectral effect Effects 0.000 description 3
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 3
- NNMALANKTSRILL-ZUTFDUMMSA-N 3-[(2z,5z)-2-[[3-(2-carboxyethyl)-5-[(z)-[(3z,4r)-3-ethylidene-4-methyl-5-oxopyrrolidin-2-ylidene]methyl]-4-methyl-1h-pyrrol-2-yl]methylidene]-5-[(4-ethyl-3-methyl-5-oxopyrrol-2-yl)methylidene]-4-methylpyrrol-3-yl]propanoic acid Chemical compound O=C1C(CC)=C(C)C(\C=C/2C(=C(CCC(O)=O)C(=C/C3=C(C(C)=C(\C=C/4\C(\[C@@H](C)C(=O)N\4)=C/C)N3)CCC(O)=O)/N\2)C)=N1 NNMALANKTSRILL-ZUTFDUMMSA-N 0.000 description 2
- GOWZVQXTHUCNSQ-NHCYSSNCSA-N Arg-Glu-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O GOWZVQXTHUCNSQ-NHCYSSNCSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 108010077544 Chromatin Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 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 2
- 108010010522 Phycobilisomes Proteins 0.000 description 2
- INPDFIMLLXXDOQ-UHFFFAOYSA-N Phycocyanobilin Natural products CCC1=C(C)C(=CC2=NC(=C/c3[nH]c(C=C/4C(C(C(N4)=O)C)=CC)c(C)c3CCC(=O)O)C(=C2C)CCC(=O)O)NC1=O INPDFIMLLXXDOQ-UHFFFAOYSA-N 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 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 2
- 238000011068 loading method Methods 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- 210000000633 nuclear envelope Anatomy 0.000 description 2
- 238000006384 oligomerization reaction Methods 0.000 description 2
- 210000002306 phycobilisome Anatomy 0.000 description 2
- 108010072011 phycocyanobilin Proteins 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 description 2
- 235000011009 potassium phosphates Nutrition 0.000 description 2
- 108010054624 red fluorescent protein Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 102200081529 rs1553259707 Human genes 0.000 description 2
- 102220123567 rs886043442 Human genes 0.000 description 2
- 210000000783 smooth endoplasmic reticulum Anatomy 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- QHASENCZLDHBGX-ONGXEEELSA-N Ala-Gly-Phe Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QHASENCZLDHBGX-ONGXEEELSA-N 0.000 description 1
- MUXONAMCEUBVGA-DCAQKATOSA-N Arg-Arg-Gln Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(N)=O)C(O)=O MUXONAMCEUBVGA-DCAQKATOSA-N 0.000 description 1
- NXDXECQFKHXHAM-HJGDQZAQSA-N Arg-Glu-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NXDXECQFKHXHAM-HJGDQZAQSA-N 0.000 description 1
- NMTANZXPDAHUKU-ULQDDVLXSA-N Arg-Tyr-Lys Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(O)=O)CC1=CC=C(O)C=C1 NMTANZXPDAHUKU-ULQDDVLXSA-N 0.000 description 1
- IBLAOXSULLECQZ-IUKAMOBKSA-N Asn-Ile-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CC(N)=O IBLAOXSULLECQZ-IUKAMOBKSA-N 0.000 description 1
- JPPLRQVZMZFOSX-UWJYBYFXSA-N Asn-Tyr-Ala Chemical compound NC(=O)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=C(O)C=C1 JPPLRQVZMZFOSX-UWJYBYFXSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 101710086766 FP protein Proteins 0.000 description 1
- LPYPANUXJGFMGV-FXQIFTODSA-N Gln-Gln-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)N)N LPYPANUXJGFMGV-FXQIFTODSA-N 0.000 description 1
- HPCOBEHVEHWREJ-DCAQKATOSA-N Gln-Lys-Glu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O HPCOBEHVEHWREJ-DCAQKATOSA-N 0.000 description 1
- FALJZCPMTGJOHX-SRVKXCTJSA-N Gln-Met-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(O)=O FALJZCPMTGJOHX-SRVKXCTJSA-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
- 239000004471 Glycine Substances 0.000 description 1
- 101001018064 Homo sapiens Lysosomal-trafficking regulator Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- HVHRPWQEQHIQJF-AVGNSLFASA-N Leu-Lys-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O HVHRPWQEQHIQJF-AVGNSLFASA-N 0.000 description 1
- QESXLSQLQHHTIX-RHYQMDGZSA-N Leu-Val-Thr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QESXLSQLQHHTIX-RHYQMDGZSA-N 0.000 description 1
- 102100033472 Lysosomal-trafficking regulator Human genes 0.000 description 1
- 241001647400 Mastigocladus laminosus Species 0.000 description 1
- 235000010703 Modiola caroliniana Nutrition 0.000 description 1
- 244000038561 Modiola caroliniana Species 0.000 description 1
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- KXUZHWXENMYOHC-QEJZJMRPSA-N Phe-Leu-Ala Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O KXUZHWXENMYOHC-QEJZJMRPSA-N 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- DZZCICYRSZASNF-FXQIFTODSA-N Pro-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 DZZCICYRSZASNF-FXQIFTODSA-N 0.000 description 1
- NFLNBHLMLYALOO-DCAQKATOSA-N Pro-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@@H]1CCCN1 NFLNBHLMLYALOO-DCAQKATOSA-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
- 239000012505 Superdex™ Substances 0.000 description 1
- SWIKDOUVROTZCW-GCJQMDKQSA-N Thr-Asn-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](C)C(=O)O)N)O SWIKDOUVROTZCW-GCJQMDKQSA-N 0.000 description 1
- VGNLMPBYWWNQFS-ZEILLAHLSA-N Thr-Thr-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N)O VGNLMPBYWWNQFS-ZEILLAHLSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- XWYUBUYQMOUFRQ-IFFSRLJSSA-N Val-Glu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N)O XWYUBUYQMOUFRQ-IFFSRLJSSA-N 0.000 description 1
- URIRWLJVWHYLET-ONGXEEELSA-N Val-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)C(C)C URIRWLJVWHYLET-ONGXEEELSA-N 0.000 description 1
- UKEVLVBHRKWECS-LSJOCFKGSA-N Val-Ile-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](C(C)C)N UKEVLVBHRKWECS-LSJOCFKGSA-N 0.000 description 1
- 241000082085 Verticillium <Phyllachorales> Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 238000005844 autocatalytic reaction Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003809 bile pigment Substances 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 108091006090 chromatin-associated proteins Proteins 0.000 description 1
- 230000001427 coherent effect Effects 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
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 108010060199 cysteinylproline Proteins 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- HJUFTIJOISQSKQ-UHFFFAOYSA-N fenoxycarb Chemical compound C1=CC(OCCNC(=O)OCC)=CC=C1OC1=CC=CC=C1 HJUFTIJOISQSKQ-UHFFFAOYSA-N 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 150000003278 haem Chemical class 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000010859 live-cell imaging Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000001273 protein sequence alignment Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0045—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Radiology & Medical Imaging (AREA)
- Epidemiology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Behavior & Ethology (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种小分子的近红外光荧光蛋白，所述近红外荧光蛋白包括BDFP近红外光荧光蛋白的氨基酸序列，并且包括在第24位，27位，30位，31位，38位的氨基酸处的突变，所述BDFP远红光荧光蛋白的氨基酸序列如SED ID NO:1～14任一所示。本发明提供的近红外荧光蛋白有效亮度高，分子量小并且是单体结构的近红外荧光蛋白，相比现有近红外荧光蛋白更适合作为蛋白融合标签序列。
Description
技术领域
本发明属于荧光标记物技术领域，更具体地，涉及一种小分子近红外光荧光蛋白及其融合蛋白。
背景技术
远红光(FR)或近红外光(NIR)在动物组织中光吸收和光散射较低，有较高的穿透性，是穿透皮肤等大部分组织的能力最大的光谱区域。具有这类发光色素基团的荧光蛋白更适宜于动物活体组织的深层成像，是活体成像更为理想的荧光标记物。
目前该类荧光标记物主要有两种，分子量大小均在35kD左右。一种源自绿色荧光蛋白(GFP),能自催化形成发色团，但是光谱范围有一定局限性，最大荧光发射波长一般在670nm左右，例如标记物TagRFP675。另一种源自存在千细菌中的受体蛋白，细菌光敏色素蛋白(BphP)。BphP主要利用线性四吡咯结构的胆绿素(BV)作为发色团；同时胆绿素BV广泛存在千真核生物体内，这意味着BphP类荧光标记物可应用于活的动物细胞和组织，且无需任何酌或外源辅助因子。BphP类标记物的代表iFP系列和iRFP系列，荧光发射波长范围为670nm-720nm，比如IFP2.0最大荧光发射波长714nm。
藻胆蛋白(phycobi1iprotein)存在远红光范围的荧光发射，机制与细菌光敏色素蛋白(BphP)类似，主要源自于非共价结合的藻蓝胆素(PCB)。典型的藻胆蛋白类荧光标记物，例如ApcA、smURFP、ApcF2，它们的最大荧光发射波长为698nm。
Ding W L等人基于藻胆蛋白体的核心亚基ApcF2的序列，进行基因改造后得到了几种新的荧光藻胆蛋白并将其命名BDFP，这些BDFP蛋白可共价结合胆绿素BV，性能比ApcF2稳定，另外，这些BDFP蛋白的分子最较小，约15kD，最大荧光发射波长为710nm左右。
虽然Ding W L等人得到的BDFP蛋白很好地弥补了现有的远红光或近红外光的荧光蛋白的上述缺点，但是这些蛋白荧光发射波长比较单一(均在710nm左右)，因此不能有效地组合使用。因此通过基因工程改造，得到亮度更高、光谱性质更为多样和优异的荧光蛋白，具有非常重要的意义。
发明内容
本发明要解决的技术问题在于克服现有技术中近红外光荧光蛋白种类不多、发射波长较为单一且亮度不够高，且蛋白分子量较大的技术不足，提供一种小分子的近红外光荧光蛋白。
本发明要解决的另一技术问题是提供一种融合荧光蛋白。
本发明还一要解决的技术问题是提供编码上述近红外光荧光蛋白或融合荧光蛋白的核酸。
本发明还一要解决的技术问题是提供一种包括上述核酸的载体。
本发明还一要解决的技术问题是提供上述红外光荧光蛋白或融合荧光蛋白在细胞荧光定位方面的应用。
本发明还一要解决的技术问题是提供上述红外光荧光蛋白或融合荧光蛋白在动物活体组织的深层成像方面的应用。
本发明的目的通过以下技术方案予以实现：
提供一种近红外光荧光蛋白，所述近红外光荧光蛋白包括BDFP近红外光荧光蛋白的氨基酸序列，并且包括在第24位，27位，30位，31位，38位的氨基酸处的突变，所述BDFP远红光荧光蛋白为以ApcF2(Chroococcidiopsis thermalis sp.PCC7203)为模板的BDFP蛋白系列。
更具体地，所述BDFP远红光荧光蛋白的序列如SEQ ID NO:1～14任一所示。
进一步地，第24位的氨基酸突变为精氨酸；第27位的氨基酸突变为谷氨酰胺；第30位的氨基酸突变为谷氨酰胺；第31位的氨基酸突变为谷氨酰胺；第38位缬氨酸突变为精氨酸。
优选地，所述第24位缬氨酸突变为精氨酸；第27位亮氨酸突变为谷氨酰胺；第30位亮氨酸突变为谷氨酰胺；第31位亮氨酸突变为谷氨酰胺；第38位缬氨酸突变为精氨酸。
更优选地，所述近红外光荧光蛋白的氨基酸序列如SEQ ID NO:15所示。
提供一种融合荧光蛋白，其所述融合荧光蛋白包括上述近红外光荧光蛋白。
提供编码上述近红外光荧光蛋白或上述融合荧光蛋白的核酸。
提供一种包括上述核酸的载体。
提供上述近红外光荧光蛋白或上述融合荧光蛋白在细胞荧光定位方面的应用。
提供上述近红外光荧光蛋白或上述融合荧光蛋白在动物活体组织的深层成像方面的应用。
本发明的有益效果是：
本发明提供了多种小分子的近红外光荧光蛋白，发射波长在694nm到705nm左右，且保留了现有BDFP1.6高亮度的特性，其中BDFP1.9，为单体结构，具有近红外光荧光蛋白中最小的分子量(17kD)，光谱略蓝移，有效亮度近似于IFP2.0。大多数的BDFPs蛋白为二聚体结构。但是在应用方面，单体结构的荧光蛋白，由于不会影响目标蛋白的化学计量，因而更适合作为蛋白融合标签序列。
本发明提供的单体结构的BDFP1.9近红外光荧光蛋白在低pH、高浓度盐酸胍溶液或高温的环境中，都具有优异的稳定性，而且也能耐受光漂白。
近红外荧光蛋白(FPs)是实现深度成像的有力工具，本发明为深度成像的荧光蛋白提供的了更多的选择，且本发明提供的近红外荧光蛋白可与其他荧光蛋白联合使用，小分子的近红外荧光蛋白更适合作为蛋白融合标签序列。
附图说明
图1BDFPs荧光蛋白的光谱和亮度比较：(a)BDFPs的吸光度和荧光光谱，蛋白样品经Ni2+亲和层析纯化；(c)相同条件下HEK 293t细胞中BDFPs与iRFP720和IFP2.0的有效亮度比较。平均近红外荧光强度归一化为平均eGFP荧光强度。误差条，SEM(n＝3，图像数量)。(d)相同条件下hek293t的有效亮度与近红外FPs分子亮度的比较。将BDFP1.7的有效亮度和分子亮度设为100％。
图2BDFP1.8的模拟结构和聚集作用分析。(a)BDFP1.8的模拟结构图，红色表示与光谱红移的氨基酸，蓝色与粉色表示与有效亮度相关的氨基酸；(b)和(c)分别为BDFP1.8在M81K和A127V处的局部结构；(d)二聚体荧光蛋白ApcE产生聚合作用的氨基酸残基位点；(e)ApcE与BDFP1.8的氨基酸序列同源性比较；(f)BDFP1.6与BDFP1.8的氨基酸序列同源性比较。
图3BDFPs荧光蛋白与BV的体外聚合作用。溶液中BDFPs BV的荧光增强。将18μMBDFPs与0.1(a),(b)1，(c)10μM BV在KPB缓冲液(包含150mM/L氯化钠，pH值7.2)中孵化，利用F＝A1-A2exp-kt方程拟合荧光指数增长。(d)HEK 293t细胞中BDFPs的有效亮度与平均k值(t50％＝ln2/k)的关系，BDFP1.7的有效亮度设为1。
图4BDFPs荧光蛋白分子大小及细胞内荧光强度。(a)BDFPs荧光蛋白经排阻色谱的结果；(b)使用的蛋白marker经排阻色谱的结果；(c)BDFPs荧光蛋白SDS-PAGE结果；(d)eGFP、mCherry、BDFPs融合荧光蛋白的质粒在HeLa细胞中表达后的荧光显微成像；(e)几种融合荧光蛋白在HeLa细胞的光滑内质网的荧光强度比。
图5BDFPs与IFP2.0、iRFP720在HEK 293T细胞中的光漂白处理中荧光的保持时间：在HEK 293T细胞中表达BDFPs、IFP2.0和iRFP720，转染24小时后检测对其进行光漂白处理，在640nm二极管激光器(最大输出功率为100mW的77％)的连续照射下，检测荧光的保持时间。
图6BDFPs与IFP2.0、iRFP720的体外稳定性比较。(a)pH2-9酸碱环境下，BDFPs与IFP2.0、iRFP720的稳定性；(b)BDFPs与IFP2.0、iRFP720与在不同浓度盐酸胍(GdnHCl)溶液(pH 7.2)中的稳定性；(c)BDFPs与IFP2.0、iRFP720在80℃高温下的稳定性；(d)BDFPs与IFP2.0、iRFP720光漂白处理中荧光的保持时间：FPs在KPB缓冲液(pH 7.2)中，100WHBO103W/2灯照射下的光漂白，BDFPs和IFP2.0采用近红外滤光片组(λex＝650/45nm和λem＝710/50nm)，iRFP720采用近红外滤光片组(λex＝650/45nm和λem＝720/40nm)，将光通过C-Apochromat油浸透镜(100×，数值孔径＝1.2)聚焦在Zeiss Axioscope A1显微镜上，该显微镜配有cool-snap HQ2CCD相机，荧光强度曲线拟合采用单指数衰减。
具体实施方式
以下结合具体的实施例与附图对本发明的技术方案做进一步说明，但本发明并不限于这些具体实施方式。实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1BDFPs突变体的载体构建、表达及性质测定
突变起始模板BDFP1.6(SEQ ID NO.1，即ApcF2(20-169)-
F30L/S46T/I51V/N72C/Y82C/Y92M/D101G/E107G/L109M/L113F/G125C/T127A/S130G/N136K/V143A/T151A/V160I/V161A/E163V)来源于藻种Chroococcidiopsisthermalis sp.PCC7203的藻胆体核亚基蛋白ApcF2。BDFP1.6，由BDFP1.1(ApcF2(20-169)-S46T/I51V/N72C/Y82C/Y92M/N136K/V160I/V161A)进化而来。BDFP1.6的荧光范围由近红外光蓝移至远红光；特别是其有效亮度甚至优于常用的iRFP670，而分子量只有其一半大小。
通过长时间创造性地研究发现，第113位，125位和127位氨基酸残基对BDFPs蛋白的光谱性质有着重要影响。BDFP1.1中的这些残基分别是亮氨酸(L)、甘氨酸(G)和苏氨酸(T)，而BDFP1.6中的苯丙氨酸(F)、半胱氨酸(C)和丙氨酸。首先对BDFP1.6的序列的113位，125位，127位氨基酸残基进行回复突变(F113L/C125G/A127T)得到BDFP1.7序列(SEQ IDNO.3)，后续基于BDFP1.7序列，对127位氨基酸残基再次突变(T127V)，得到的V6序列(SEQID NO.4)。进一步研究哪些氨基酸残基可能增强色素基团与脱辅基蛋白亲和力，后续在V6序列突变体的基础上，再进一步针对相关氨基酸进行突变，得到V7～V18的序列。另一方面，采用串联方案设计的单体结构的BDFP，以ApcE为模板进行对比分析，根据分析比对，认定第24位，27位，30位，31位，38位氨基酸残基对聚集作用有影响，可在BDFP序列基础上进行突变，通过创造性地研究发现，将BDFP第24位的氨基酸突变为精氨酸；第27位的氨基酸突变为谷氨酰胺；第30位的氨基酸突变为谷氨酰胺；第31位的氨基酸突变为谷氨酰胺；第38位的氨基酸突变为精氨酸后，可以改变BDFP的聚集作用，得到一系列小分子单体荧光蛋白，其中，在BDFP1.8序列基础上，通过将第24位缬氨酸突变为精氨酸；第27位亮氨酸突变为谷氨酰胺；第30位亮氨酸突变为谷氨酰胺；第31位亮氨酸突变为谷氨酰胺；第38位缬氨酸突变为精氨酸改造得到的BDFP1.9在这一系列的小分子单体荧光蛋白中具有最高的有效亮度。具体序列名称、突变位点、对应序列序号见下表1。
表1蛋白/突变体名称、突变位点及编号
1.克隆及融合体构建
pET28和pACYCDuet(Novagen)是T7启动子表达载体。pACYCDuet经设计，能够用于双目的基因序列在大肠杆菌(E.coli)内共同转化表达。
荧光蛋白序列的编码基因可通过酶切位点NcoI和XhoI，克隆进入pET28a载体。血红素氧化酶基因ho1可克隆进入pACYCDuet载体质粒，用于产生胆绿素BV。
表达载体pcDNA3.1(Invitrogen)是带有CMV启动子的哺乳动物类表达载体。
在HEK 293T细胞内进行筛选时，使用表达载体pcDNA3.1，设计融合表达序列为FP:IRES:eGFP。作亮度对比时，可基于eGFP的荧光亮度，对FP蛋白的荧光亮度进行修正。
二倍串联融合蛋白设计(BDFP1.7:1.7，BDFP1.8:1.8等)，采用的连接序列(linker)为11个氨基酸残基的连接子：GHGTGSTGSGS(SEQ ID NO.17)。采用一步克隆的方法构建了BDFP 1.7:1.7和BDFP 1.8:1.8的DNA。
2.大肠杆菌表达
将带有编码荧光蛋白的pET表达载体转化到大肠杆菌菌株BL21(DE3)(Novagen)后，再将载体pACYC-ho1转化到同一菌株中。将经转化的BL21细胞在18℃，培养在补充有卡那霉素(20μg/ml)和氯霉素(17μg/ml)LB培养基中。当O.D值达到0.4～0.6时，使用1mM异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达5～16小时，然后在4℃，12,000×g离心3min，收集细胞，经水冲洗2次，短期保存在4℃或者置于-20℃长期保存。
3.哺乳动物细胞表达
将HEK 293T或HeLa细胞培养在含10％胎牛血清的DMEM培养基(Invitrogen)中。使用
3000(Invitrogen)进行转染。转染时，
2000与DNA以2:1(μl:μg)的比率在无血清培养基Opti-
中混合10分钟后，立即加入到要转染的细胞中。6～8h后，更换新鲜的DMEM培养基。
荧光蛋白表达24h后开始进行活体细胞成像(镜检)。成像前先用1mL PBS冲洗两次，然后用DEME培养基(无酚红)冲洗一次。成像设备为倒置显微镜Nikon Ti，配有cool-snap HQ2CCD相机和Nikon Plan Fluor ELWD 20×0.45-DIC L-WD物镜。激发和发射设置如下：eGFP为绿色通道，λex＝470/40，λem＝510/40nm；近红外光BDFPs蛋白和IFP2.0为近红外光通道1，λex＝650/40，λem＝710/50nm；iRFP720为近红外光通道2，λex＝650/40，λem＝720/40nm。图片使用ImageJ软件(National Institutes of Health)进行分析和处理。
4.蛋白纯化与定量
将湿菌体悬浮在冰预冷的起始缓冲液[磷酸钾缓冲液(KPB,20mM,pH 7.2)，氯化钠(NaCl,0.5M)]中。经50W功率超声(JY92-II,宁波新芝生物科技股份有限公司，中国)破碎，5分钟。悬浮液在4℃，12000×g离心60分钟。得到的上清液经Ni2+亲和层析柱(AmershamBiosciences)纯化，其中使用起始缓冲液[磷酸钾(KPB,20mM,pH 7.2)上样，使用额外含有0.5M咪唑的缓冲液进行洗脱。收集到的样品，用起始缓冲液(pH 7.2)透析至少两次。通过Bradford法测定蛋白浓度，使用牛血清白蛋白作为标准品进行校正。纯化后的蛋白经排阻色谱以及SDS-PAGE验证蛋白分子大小。用Zn2+诱导的荧光法对其染色，然后用考马斯亮蓝染色，结果如附图4(c)所示。
5.同源建模分析
BDFP荧光蛋白结构比对，在SWISS-MODEL远程服务器上完成。采用的模板序列为层理鞭枝藻(Mastigocladuslaminosus)的藻胆蛋白ApcB(pdb code:1B33)，对比软件为Swiss-PDB Viewer，版本4.1。采用PyMOL(http://www.pymol.org/)创建蛋白结构图。采用Clustal(http://www.clustal.org/)创建完成蛋白序列比对图。
6.蛋白寡聚状态分析
采用分子筛纯化法，通过与一组蛋白质Marker(12-66kDa；Sigma-Aldrich)进行对比，可测定蛋白样品的分子量大小，进而推算其寡聚状态。蛋白样品上样量为1mL，样品经过Ni2+亲和层析纯化，并透析至KPB缓冲液(20mM,pH7.2,含150mM NaCl)条件下。分子筛柱型为Superdex 75(30×1.0cm)，洗脱缓冲液条件同样品。
7.光谱分析
将Ni2+亲和层析纯化的荧光蛋白进行吸收光谱检测，是通过UV-9000S分光光度计(Shanghai Metash Instruments Co.,Ltd)来完成。
荧光蛋白的消光系数是依据胆色素BV在390nm处的吸收系数ε＝39,900M-1cm-1，进行参比换算的。
荧光光谱是通过荧光分光光度计(F320，天津港东科技发展股份有限公司)来检测完成。参考BDFP1.1荧光蛋白(ΦF＝0.059)检测荧光量子产率ΦF，样品检测环境为磷酸钾盐溶液(20mM,pH 7.2，KPB)。
8.广域与超分辨率显微镜成像
在室温，通过独立模式，使用配有100×1.49NA油浸物镜的ECLIPSE Ti-E倒置尼康显微镜上的尼康结构化照明系统，获取广域和结构化照明显微镜(SIM)照片。使用640nm的半导体激光器(100mW,CUBE 640-100C,COHERENT)激发NIR荧光。使用受NIS-Elements AR软件(尼康)控制的电子倍增CCD相机(Andor iXon3DU897)进行数据采集。使用NIS-ElementsAR对图像进行处理。
9.定量与统计分析
所有的荧光照片都统一使用工具软件ImageJ(National Institutes of Health)进行调校分析。数据图和统计使用工具软件Origin 8.0(OriginLab)。
表2不同近红外光荧光蛋白(NIR FRs)的性质对比
从结果中可以看出，BDFP1.7蛋白，在680nm处吸收最多，在705nm处发出荧光。虽然BDFP1.7的分子亮度仅比BDFP1.1高1.09倍，但在HEK 293T细胞中，BDFP1.7的有效亮度比BDFP1.1高6.25倍。基于BDFP1.7序列，对127位氨基酸残基再次突变(T127V)得到的V6突变体，显著提高了BDFP1.7的有效亮度2.3倍。模型结构表明，V6突变体中，两个甲基可能作为“钳”作用，通过范德瓦尔斯相互作用锁住BV染色质环，增强荧光，如附图2(c)所示。另一个突变(M81K)可能会在染色质和脱辅基蛋白之间产生氢键，如附图2(b)所示，所以加入了这个突变，使得有效亮度比V6突变体提高了1.4倍。
游离状态的BV是非荧光的，在体外，BDFPs与BV的结合需要三个动力学步骤，第一步，BDFP1.1、1.7、V6、1.8和脱辅基蛋白蛋白非共价结合BV，产生长波长发射(～712nm)。随后，光谱发生蓝移，荧光增强。BDFP1.2和1.6的蓝移为～40nm，而BDFP1.1、1.7、V6突变体和1.8的蓝移仅为5-10nm。在第二步中，这种变化可能是由于在BV 3-乙烯基上添加了C82巯基导致共价附着，从而使荧光发生蓝移，如附图2(a)所示。此外，荧光的增加可以通过第三步共价染色质结合后的进一步重组来解释。有趣的是，BDFP1.8和V6突变体与BV组装的速度要快于其他两个，如附图3所示；因此推测范德瓦尔斯相互作用和氢键可以显著加快BV组装的速度。此外，有效亮度与BV组装率的线性关系表明，BDFPs与BV组装越快，哺乳动物细胞的有效亮度越高，如附图3所示。有效亮度与分子亮度明显无关，如附图1(d)所示。
经过上述分子进化，得到的BDFP1.8(Fmax＝702nm)的近红外光荧光蛋白，其在HEK293T细胞中的有效亮度分别是BDFP1.1和BDFP1.7的20.25和3.24倍。此外，BDFP1.8有效亮度高于iRFP720的2.4倍，这是报告为最亮的与BV组装的近红外光荧光蛋白组装(见f附图1(c)和表2)。且BDFP1.8只有iRFP720分子质量的一半，相比于iRFP720，BDFP1.8应该是更优的荧光生物标记。
经过对BDFP1.8改造得到的BDFP1.9，为单体结构，最小分子量(17kD)，光谱略蓝移，有效亮度近似于IFP2.0，但分子量只有IFP2.0分子质量的一半，而且为单体的荧光蛋白。
用HeLa细胞检测BDFPs的低聚状态。转染和活细胞成像如上所述。选择二聚体荧光蛋白(eGFP)作为阳性对照，测定有组织的光滑内质网(OSER)结构；mCherry被报道为一种真正的单体荧光蛋白，作为阴性对照。利用ImageJ(美国国立卫生研究院)计算了OSER结构的平均强度与三个核膜(NE)区域的平均强度之比。在没有可见OSER结构的情况下，采用点状非OSER结构，结果见下表3及附图4。从结果中可以看出，BDFP1.9在细胞中呈紫红色荧光，由于eGFP为二聚体结构，在细胞中组织定位时会有明显聚集作用，即为附图4中膜结构中的亮点，因信号过强反而可能存在显示不清细胞结构的情况，而单体结构的mCherry荧光蛋白则不会有聚集的亮点，也进一步佐证了BDFP1.9蛋白与mCherry荧光蛋白呈单体结构。
表3突变体序列(相比BDFP1.6)及其有效亮度(相比BDFP1.7)对比
HeLa细胞中表达eGFP、mCherry、BDFPs荧光蛋白，荧光显微成像见附图4(d)，对应的荧光强度比见附图4(e)。
实施例2BDFPs突变体荧光蛋白的稳定性
本实施例检测了荧光蛋白对体外条件，例如酸碱、变性条件、高温、光漂白的耐受性。结果示于图6中。
图6(a)中展示了pH2-9酸碱环境下，BDFPs以及IFP2.0和iRFP720的稳定性，BDFPs的荧光强度在pH从9到2的范围内仅变化40％，而IFP2.0在pH3.5时、iRFP720在pH2时荧光已经完全猝灭了。这说明BDFP1.9在低pH下是相对稳定的，在酸性条件下，具有比IFP2.0和iRFP720更高的稳定性。
图6(b)中展示了BDFPs以及IFP2.0和iRFP720在不同浓度的盐酸胍溶液中的荧光强度。从图中可以看出，IFP2.0、iRFP720和BDFP1.1在4M的盐酸胍溶液中荧光已经完全猝灭了，而BDFP1.9仍保留30％以上的荧光，说明BDFP1.9在高浓度变性剂条件下，相比IFP2.0和iRFP720具有更好的稳定性。
图6(c)中展示了BDFPs以及IFP2.0和iRFP720在80℃条件下荧光保持的时间。从图中可以看出，相比IFP2.0和iRFP720，BDFP1.9在80℃温浴2h后，仍保留有40％以上的荧光。而IFP2.0在80℃的高温下，10分钟就已经猝灭，iRFP720在80℃温浴2h后，荧光强度也降低到10％以下。
图6(d)中展示了BDFPs以及IFP2.0和iRFP720在光漂白处理中荧光的保持时间。从图中可以看出，BDFP1.9在光漂白处理中荧光的保持时间也远比IFP2.0和iRFP720长。
基于上述结果，可以得出经检测，BDF,1.9在低pH、高浓度盐酸胍溶液或高温的环境中，都具有优异的稳定性，而且也能耐受光漂白，相比IFP2.0和iRFP720具有跟高的稳定性。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。
SEQUENCE LISTING
<110> 华中农业大学
广州天宝颂原生物科技开发有限公司
<120> 一种小分子近红外光荧光蛋白及其融合蛋白
<130>
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 152
<212> PRT
<213> 人工合成
<400> 1
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Phe
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Cys Pro Ala Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 2
<211> 152
<212> PRT
<213> 人工合成
<400> 2
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Leu
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Thr Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 3
<211> 152
<212> PRT
<213> 人工合成
<400> 3
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Leu
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Val Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 4
<211> 152
<212> PRT
<213> 人工合成
<400> 4
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Phe Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Leu
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Val Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 5
<211> 152
<212> PRT
<213> 人工合成
<400> 5
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr His Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Leu
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Val Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 6
<211> 152
<212> PRT
<213> 人工合成
<400> 6
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Lys Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Leu
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Val Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 7
<211> 152
<212> PRT
<213> 人工合成
<400> 7
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Lys
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Leu
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Val Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 8
<211> 152
<212> PRT
<213> 人工合成
<400> 8
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Asn Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Leu
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Val Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 9
<211> 152
<212> PRT
<213> 人工合成
<400> 9
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Asp Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Leu
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Val Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 10
<211> 152
<212> PRT
<213> 人工合成
<400> 10
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Gln Met Leu Ala Gly Leu
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Val Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 11
<211> 152
<212> PRT
<213> 人工合成
<400> 11
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Leu
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Val Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Val Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 12
<211> 152
<212> PRT
<213> 人工合成
<400> 12
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Leu
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Val Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Thr Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 13
<211> 152
<212> PRT
<213> 人工合成
<400> 13
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Val Val Val Lys Ser Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Leu
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Val Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 14
<211> 152
<212> PRT
<213> 人工合成
<400> 14
Met Ala Asn Arg Glu Val Val Glu Thr Leu Lys Glu Leu Leu Ala Asp
1 5 10 15
Gly Glu Lys Arg Val Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Gly Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Ala Arg Arg Tyr Asn Met
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Leu
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Val Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
<210> 15
<211> 152
<212> PRT
<213> 人工合成
<400> 15
Met Ala Asn Arg Glu Val Arg Glu Thr Gln Lys Glu Gln Gln Ala Asp
1 5 10 15
Gly Glu Lys Arg Arg Gln Val Ala Gly Val Ile Gly Thr Asn Ala Ala
20 25 30
Glu Val Val Lys Thr Ala Val Ser Leu Leu Phe Gln Glu Tyr Pro Glu
35 40 45
Leu Val Ser Pro Gly Gly Cys Ala Tyr Thr Thr Arg Arg Tyr Asn Lys
50 55 60
Cys Val Arg Asp Met Asn Tyr Phe Leu Arg Met Cys Ser Tyr Ala Ile
65 70 75 80
Val Ala Gly Gly Ala Ser Val Leu Asp Gly Arg Met Leu Ala Gly Leu
85 90 95
Arg Asp Thr Phe Asn Ser Leu Gly Ile Pro Leu Gly Pro Val Ala Arg
100 105 110
Gly Ile Gln Leu Met Lys Lys Ile Val Lys Glu Lys Leu Ala Thr Ala
115 120 125
Gly Met Thr Asn Ile Ala Phe Val Asp Glu Pro Phe Asp Tyr Ile Ala
130 135 140
Arg Val Ile Ser Glu Thr Glu Ile
145 150
Claims (8)
1.一种近红外光荧光蛋白，所述近红外光荧光蛋白的氨基酸序列如SEQ ID NO:15所示。
2.一种融合荧光蛋白，其特征在于，所述融合荧光蛋白包括权利要求1所述近红外光荧光蛋白。
3.编码权利要求1所述近红外光荧光蛋白的核酸。
4.编码权利要求2所述融合荧光蛋白的的核酸。
5.一种载体，所述载体中含有权利要求3所述的核酸或权利要求4所述的核酸。
6.一种细胞，所述细胞中含有权利要求3所述的核酸，所述细胞为非植物或动物品种。
7.权利要求1所述的近红外光荧光蛋白或权利要求2所述的融合荧光蛋白在细胞荧光定位方面的应用。
8.权利要求1所述的近红外光荧光蛋白或权利要求2所述的融合荧光蛋白在动物活体组织的深层成像方面的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910586801.3A CN110577593B (zh) | 2019-07-01 | 2019-07-01 | 一种小分子近红外光荧光蛋白及其融合蛋白 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910586801.3A CN110577593B (zh) | 2019-07-01 | 2019-07-01 | 一种小分子近红外光荧光蛋白及其融合蛋白 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110577593A CN110577593A (zh) | 2019-12-17 |
| CN110577593B true CN110577593B (zh) | 2021-09-14 |
Family
ID=68811001
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910586801.3A Active CN110577593B (zh) | 2019-07-01 | 2019-07-01 | 一种小分子近红外光荧光蛋白及其融合蛋白 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110577593B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110386977B (zh) * | 2019-07-01 | 2022-12-13 | 广州天宝颂原生物科技开发有限公司 | 一种近红外光荧光蛋白及其融合蛋白 |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101759793A (zh) * | 2008-12-04 | 2010-06-30 | 广州天宝颂原生物科技开发有限公司 | 结合藻红胆素PEB的藻红蓝蛋白α亚基类荧光蛋白及其应用 |
| CN101759797A (zh) * | 2008-12-04 | 2010-06-30 | 广州天宝颂原生物科技开发有限公司 | 结合藻红胆素PEB的藻蓝蛋白β亚基类荧光蛋白及其应用 |
| CN101759785A (zh) * | 2008-12-04 | 2010-06-30 | 广州天宝颂原生物科技开发有限公司 | 结合藻红胆素PEB的藻蓝蛋白a亚基类荧光蛋白及其应用 |
| CN101759786A (zh) * | 2008-12-04 | 2010-06-30 | 广州天宝颂原生物科技开发有限公司 | 结合藻蓝胆素PCB的藻蓝蛋白α亚基类荧光蛋白及其应用 |
| WO2010085844A1 (en) * | 2009-01-29 | 2010-08-05 | Commonwealth Scientific Industrial Research Organisation | Measuring g protein coupled receptor activation |
| WO2012055409A1 (en) * | 2010-10-27 | 2012-05-03 | Quantibact A/S | Capture of methylated rna and/or dna sequences by specific probes |
| CN109134644A (zh) * | 2018-08-30 | 2019-01-04 | 华中农业大学 | 远红光荧光蛋白及其融合蛋白 |
| CN109265523A (zh) * | 2018-08-30 | 2019-01-25 | 广州天宝颂原生物科技开发有限公司 | 源自bdfp近红外光荧光蛋白的新型荧光标记物及其融合蛋白 |
| CN109517057A (zh) * | 2018-08-30 | 2019-03-26 | 广州天宝颂原生物科技开发有限公司 | 经基因改造的新型bdfp荧光蛋白及其融合蛋白 |
-
2019
- 2019-07-01 CN CN201910586801.3A patent/CN110577593B/zh active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101759793A (zh) * | 2008-12-04 | 2010-06-30 | 广州天宝颂原生物科技开发有限公司 | 结合藻红胆素PEB的藻红蓝蛋白α亚基类荧光蛋白及其应用 |
| CN101759797A (zh) * | 2008-12-04 | 2010-06-30 | 广州天宝颂原生物科技开发有限公司 | 结合藻红胆素PEB的藻蓝蛋白β亚基类荧光蛋白及其应用 |
| CN101759785A (zh) * | 2008-12-04 | 2010-06-30 | 广州天宝颂原生物科技开发有限公司 | 结合藻红胆素PEB的藻蓝蛋白a亚基类荧光蛋白及其应用 |
| CN101759786A (zh) * | 2008-12-04 | 2010-06-30 | 广州天宝颂原生物科技开发有限公司 | 结合藻蓝胆素PCB的藻蓝蛋白α亚基类荧光蛋白及其应用 |
| WO2010085844A1 (en) * | 2009-01-29 | 2010-08-05 | Commonwealth Scientific Industrial Research Organisation | Measuring g protein coupled receptor activation |
| WO2012055409A1 (en) * | 2010-10-27 | 2012-05-03 | Quantibact A/S | Capture of methylated rna and/or dna sequences by specific probes |
| CN109134644A (zh) * | 2018-08-30 | 2019-01-04 | 华中农业大学 | 远红光荧光蛋白及其融合蛋白 |
| CN109265523A (zh) * | 2018-08-30 | 2019-01-25 | 广州天宝颂原生物科技开发有限公司 | 源自bdfp近红外光荧光蛋白的新型荧光标记物及其融合蛋白 |
| CN109517057A (zh) * | 2018-08-30 | 2019-03-26 | 广州天宝颂原生物科技开发有限公司 | 经基因改造的新型bdfp荧光蛋白及其融合蛋白 |
Non-Patent Citations (3)
| Title |
|---|
| Small monomeric and highly stable near-infrared fluorescent markers derived from the thermophilic phycobiliprotein, ApcF2;Wen-Long Ding等;《Biochimica et Biophysica Acta (BBA) - Molecular Cell Research》;ELSEVIER;20171031;第1864卷(第10期);第1877-1886页 * |
| 蓝藻红色荧光蛋白All1280 GAF2在E. coli BL21（DE3）中的表达及其突变体构建;马琼等;《植物科学学报》;CNKI;20090415;第37卷(第2期);第181-187页 * |
| 远红光诱导的别藻蓝蛋白F亚基分子进化为近红外荧光探针;侯亚男等;《中国化学会第四届卟啉与酞菁学术研讨会论文集》;CNKI;20170706;第50页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110577593A (zh) | 2019-12-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110386977B (zh) | 一种近红外光荧光蛋白及其融合蛋白 | |
| Gurskaya et al. | A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants | |
| Shkrob et al. | Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equina | |
| Ding et al. | Small monomeric and highly stable near-infrared fluorescent markers derived from the thermophilic phycobiliprotein, ApcF2 | |
| CN109134644B (zh) | 远红光荧光蛋白及其融合蛋白 | |
| ES2399563T3 (es) | Proteínas verdes fluorescentes modificadas y método para la utilización de las mismas | |
| JP5570803B2 (ja) | 蛍光タンパク質およびｐＨ測定方法 | |
| US20130344591A1 (en) | Modified Fluorescent Proteins and Methods for Using Same | |
| Ding et al. | Far-red acclimating cyanobacterium as versatile source for bright fluorescent biomarkers | |
| CN109553689B (zh) | 一种含有ApcE2突变体的融合蛋白及其应用 | |
| Hunt et al. | Multi-domain GFP-like proteins from two species of marine hydrozoans | |
| CN110577593B (zh) | 一种小分子近红外光荧光蛋白及其融合蛋白 | |
| Li et al. | Design of small monomeric and highly bright near-infrared fluorescent proteins | |
| CN109265523B (zh) | 源自bdfp近红外光荧光蛋白的新型荧光标记物及其融合蛋白 | |
| US8679749B2 (en) | Red fluorescent proteins with enhanced bacterial expression, increased brightness and reduced aggregation | |
| US11021523B2 (en) | Cyanobacteriochromes active in the far-red to near-infrared | |
| CN109517057B (zh) | 经基因改造的新型bdfp荧光蛋白及其融合蛋白 | |
| Hou et al. | Bright near-infrared fluorescence bio-labeling with a biliprotein triad | |
| CN109553661B (zh) | ApcE2蛋白突变体及其应用 | |
| CN102080068B (zh) | 荧光素酶活性片段及其应用 | |
| CN110305199B (zh) | 结合藻红胆素的荧光生物标记物及标记方法 | |
| US20180201655A1 (en) | ALLOPHYCOCYANIN ALPHA-SUBUNIT EVOLVED LABELING PROTEINS (smURFPs) | |
| Pletneva et al. | Structure of a red fluorescent protein from Zoanthus, zRFP574, reveals a novel chromophore | |
| CN112961225B (zh) | 近红外荧光蛋白、重组载体、重组细胞及其应用 | |
| Tu et al. | A naturally enhanced green fluorescent protein from magnificent sea anemone (Heteractis magnifica) and its functional analysis |
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 |