CN112501140A - Bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, and preparation method and application thereof - Google Patents
Bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, and preparation method and application thereof Download PDFInfo
- Publication number
- CN112501140A CN112501140A CN202011485267.6A CN202011485267A CN112501140A CN 112501140 A CN112501140 A CN 112501140A CN 202011485267 A CN202011485267 A CN 202011485267A CN 112501140 A CN112501140 A CN 112501140A
- Authority
- CN
- China
- Prior art keywords
- yfgsgfaapffivrhqllkk
- biologically active
- polypeptide
- phe
- bioactive
- 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
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 106
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 70
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 51
- 230000000975 bioactive effect Effects 0.000 title claims abstract description 50
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 claims abstract description 20
- 210000004698 lymphocyte Anatomy 0.000 claims abstract description 19
- 235000013305 food Nutrition 0.000 claims abstract description 15
- 239000003814 drug Substances 0.000 claims abstract description 12
- 210000002540 macrophage Anatomy 0.000 claims abstract description 12
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 11
- 230000035755 proliferation Effects 0.000 claims abstract description 11
- 230000007365 immunoregulation Effects 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims description 19
- 230000003110 anti-inflammatory effect Effects 0.000 claims description 15
- 210000004027 cell Anatomy 0.000 claims description 11
- 150000001413 amino acids Chemical class 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 8
- 230000002519 immonomodulatory effect Effects 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 230000001737 promoting effect Effects 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 239000002537 cosmetic Substances 0.000 claims description 4
- 230000004048 modification Effects 0.000 claims description 4
- 238000012986 modification Methods 0.000 claims description 4
- 238000010353 genetic engineering Methods 0.000 claims description 3
- 230000021736 acetylation Effects 0.000 claims description 2
- 238000006640 acetylation reaction Methods 0.000 claims description 2
- 239000002260 anti-inflammatory agent Substances 0.000 claims description 2
- 229940124599 anti-inflammatory drug Drugs 0.000 claims description 2
- 230000006315 carbonylation Effects 0.000 claims description 2
- 238000005810 carbonylation reaction Methods 0.000 claims description 2
- 230000021523 carboxylation Effects 0.000 claims description 2
- 238000006473 carboxylation reaction Methods 0.000 claims description 2
- 230000032050 esterification Effects 0.000 claims description 2
- 238000005886 esterification reaction Methods 0.000 claims description 2
- 230000013595 glycosylation Effects 0.000 claims description 2
- 238000006206 glycosylation reaction Methods 0.000 claims description 2
- 230000033444 hydroxylation Effects 0.000 claims description 2
- 238000005805 hydroxylation reaction Methods 0.000 claims description 2
- 230000011987 methylation Effects 0.000 claims description 2
- 238000007069 methylation reaction Methods 0.000 claims description 2
- 230000026731 phosphorylation Effects 0.000 claims description 2
- 238000006366 phosphorylation reaction Methods 0.000 claims description 2
- 108091033319 polynucleotide Proteins 0.000 claims description 2
- 239000002157 polynucleotide Substances 0.000 claims description 2
- 102000040430 polynucleotide Human genes 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 210000004899 c-terminal region Anatomy 0.000 claims 1
- 230000008975 immunomodulatory function Effects 0.000 claims 1
- 238000002955 isolation Methods 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 abstract description 10
- 108090000623 proteins and genes Proteins 0.000 abstract description 10
- 238000002474 experimental method Methods 0.000 abstract description 6
- 238000000338 in vitro Methods 0.000 abstract description 6
- 229940079593 drug Drugs 0.000 abstract description 3
- 230000006698 induction Effects 0.000 abstract description 3
- 208000015181 infectious disease Diseases 0.000 abstract description 3
- 244000052769 pathogen Species 0.000 abstract description 3
- 230000036737 immune function Effects 0.000 abstract description 2
- 230000001105 regulatory effect Effects 0.000 abstract 2
- 239000011347 resin Substances 0.000 description 21
- 229920005989 resin Polymers 0.000 description 21
- 230000006870 function Effects 0.000 description 16
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 13
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 12
- 102100030512 Cytochrome c oxidase subunit 7C, mitochondrial Human genes 0.000 description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 9
- 101000919491 Homo sapiens Cytochrome c oxidase subunit 7C, mitochondrial Proteins 0.000 description 8
- 239000013642 negative control Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- 102000006404 Mitochondrial Proteins Human genes 0.000 description 6
- 108010058682 Mitochondrial Proteins Proteins 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 230000004957 immunoregulator effect Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 210000000952 spleen Anatomy 0.000 description 5
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 206010061218 Inflammation Diseases 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 238000010511 deprotection reaction Methods 0.000 description 4
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 4
- 230000004054 inflammatory process Effects 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 230000000638 stimulation Effects 0.000 description 4
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 108010077544 Chromatin Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 206010057249 Phagocytosis Diseases 0.000 description 3
- 210000003483 chromatin Anatomy 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 230000008782 phagocytosis Effects 0.000 description 3
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 3
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 2
- HBOMLICNUCNMMY-KJFJCRTCSA-N 1-[(4s,5s)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1C1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-KJFJCRTCSA-N 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000003226 decolorizating effect Effects 0.000 description 2
- BGRWYRAHAFMIBJ-UHFFFAOYSA-N diisopropylcarbodiimide Natural products CC(C)NC(=O)NC(C)C BGRWYRAHAFMIBJ-UHFFFAOYSA-N 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- USSYUMHVHQSYNA-SLDJZXPVSA-N indolicidin Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(N)=O)CC1=CNC2=CC=CC=C12 USSYUMHVHQSYNA-SLDJZXPVSA-N 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 210000003024 peritoneal macrophage Anatomy 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- ZQOILFFBJUNGRA-NMVUUJPQSA-N (4s)-4-[[(2s)-2-amino-3-methylbutanoyl]amino]-5-[(2s)-2-[[(2s,3s)-1-[(2s)-2-[[(1s)-1-carboxy-2-(4-hydroxyphenyl)ethyl]carbamoyl]pyrrolidin-1-yl]-3-methyl-1-oxopentan-2-yl]carbamoyl]pyrrolidin-1-yl]-5-oxopentanoic acid Chemical compound N([C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C(=O)[C@@H]1CCCN1C(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)C(C)C ZQOILFFBJUNGRA-NMVUUJPQSA-N 0.000 description 1
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 description 1
- 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 1
- VEAPAYQQLSEKEM-GUBZILKMSA-N Ala-Met-Met Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCSC)C(O)=O VEAPAYQQLSEKEM-GUBZILKMSA-N 0.000 description 1
- JEXPNDORFYHJTM-IHRRRGAJSA-N Arg-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCCN=C(N)N JEXPNDORFYHJTM-IHRRRGAJSA-N 0.000 description 1
- PRLPSDIHSRITSF-UNQGMJICSA-N Arg-Phe-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PRLPSDIHSRITSF-UNQGMJICSA-N 0.000 description 1
- JLNFZLNDHONLND-GARJFASQSA-N Asn-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N JLNFZLNDHONLND-GARJFASQSA-N 0.000 description 1
- XFJKRRCWLTZIQA-XIRDDKMYSA-N Asn-Lys-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(=O)N)N XFJKRRCWLTZIQA-XIRDDKMYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 101710090949 Cytochrome c oxidase subunit 7C, mitochondrial Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 238000012270 DNA recombination Methods 0.000 description 1
- SXFPZRRVWSUYII-KBIXCLLPSA-N Gln-Ser-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)N)N SXFPZRRVWSUYII-KBIXCLLPSA-N 0.000 description 1
- SJPMNHCEWPTRBR-BQBZGAKWSA-N Glu-Glu-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O SJPMNHCEWPTRBR-BQBZGAKWSA-N 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 241000588747 Klebsiella pneumoniae Species 0.000 description 1
- 241000209499 Lemna Species 0.000 description 1
- HZVXPUHLTZRQEL-UWVGGRQHSA-N Met-Leu-Gly Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O HZVXPUHLTZRQEL-UWVGGRQHSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 102000001490 Opioid Peptides Human genes 0.000 description 1
- 108010093625 Opioid Peptides Proteins 0.000 description 1
- 208000025174 PANDAS Diseases 0.000 description 1
- 208000021155 Paediatric autoimmune neuropsychiatric disorders associated with streptococcal infection Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- BIYWZVCPZIFGPY-QWRGUYRKSA-N Phe-Gly-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](CO)C(O)=O BIYWZVCPZIFGPY-QWRGUYRKSA-N 0.000 description 1
- FENSZYFJQOFSQR-FIRPJDEBSA-N Phe-Phe-Ile Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FENSZYFJQOFSQR-FIRPJDEBSA-N 0.000 description 1
- MVIJMIZJPHQGEN-IHRRRGAJSA-N Phe-Ser-Val Chemical compound CC(C)[C@@H](C([O-])=O)NC(=O)[C@H](CO)NC(=O)[C@@H]([NH3+])CC1=CC=CC=C1 MVIJMIZJPHQGEN-IHRRRGAJSA-N 0.000 description 1
- UIMCLYYSUCIUJM-UWVGGRQHSA-N Pro-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 UIMCLYYSUCIUJM-UWVGGRQHSA-N 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- JFWDJFULOLKQFY-QWRGUYRKSA-N Ser-Gly-Phe Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JFWDJFULOLKQFY-QWRGUYRKSA-N 0.000 description 1
- WEQAYODCJHZSJZ-KKUMJFAQSA-N Ser-His-Tyr Chemical compound C([C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CN=CN1 WEQAYODCJHZSJZ-KKUMJFAQSA-N 0.000 description 1
- IEZVHOULSUULHD-XGEHTFHBSA-N Thr-Ser-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O IEZVHOULSUULHD-XGEHTFHBSA-N 0.000 description 1
- BPGDJSUFQKWUBK-KJEVXHAQSA-N Thr-Val-Tyr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 BPGDJSUFQKWUBK-KJEVXHAQSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- OKDNSNWJEXAMSU-IRXDYDNUSA-N Tyr-Phe-Gly Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(O)=O)C1=CC=C(O)C=C1 OKDNSNWJEXAMSU-IRXDYDNUSA-N 0.000 description 1
- WGHVMKFREWGCGR-SRVKXCTJSA-N Val-Arg-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N WGHVMKFREWGCGR-SRVKXCTJSA-N 0.000 description 1
- HNWQUBBOBKSFQV-AVGNSLFASA-N Val-Arg-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N HNWQUBBOBKSFQV-AVGNSLFASA-N 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000003178 anti-diabetic effect Effects 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000004807 desolvation Methods 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 235000021244 human milk protein Nutrition 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 235000019239 indanthrene blue RS Nutrition 0.000 description 1
- UHOKSCJSTAHBSO-UHFFFAOYSA-N indanthrone blue Chemical compound C1=CC=C2C(=O)C3=CC=C4NC5=C6C(=O)C7=CC=CC=C7C(=O)C6=CC=C5NC4=C3C(=O)C2=C1 UHOKSCJSTAHBSO-UHFFFAOYSA-N 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000003399 opiate peptide Substances 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- YYVGYULIMDRZMJ-UHFFFAOYSA-N propan-2-ylsilane Chemical compound CC(C)[SiH3] YYVGYULIMDRZMJ-UHFFFAOYSA-N 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000006433 tumor necrosis factor production Effects 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 108010030651 valyl-glutamyl-prolyl-isoleucyl-prolyl-tyrosine Proteins 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0053—Oxidoreductases (1.) acting on a heme group of donors (1.9)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/66—Enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y109/00—Oxidoreductases acting on a heme group of donors (1.9)
- C12Y109/03—Oxidoreductases acting on a heme group of donors (1.9) with oxygen as acceptor (1.9.3)
- C12Y109/03001—Cytochrome-c oxidase (1.9.3.1)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention relates to the field of protein, and in particular relates to a bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, a preparation method and application thereof, wherein the amino acid sequence of the bioactive polypeptide YFGSGFAAPFFIVRHQLLKK is Tyr-Phe-Gly-Ser-Gly-Phe-Ala-Ala-Pro-Phe-Phe-Ile-Val-Arg-His-Gln-Leu-Leu-Lys-Lys. In vitro immune function regulating experiment proves that the polypeptide YFGSGFAAPFFIVRHQLLKK has better immune regulating function. The bioactive peptide YFGSGFAAPFFIVRHQLLKK can promote the increase of the induction quantity of macrophage nitric oxide, improve the capability of an organism for resisting the infection of external pathogens, reduce the morbidity of the organism, obviously promote the proliferation of mouse lymphocytes, improve the quality of life and have very important significance for developing foods, health-care products and medicines with the immunoregulation function.
Description
Technical Field
The invention relates to the field of proteins, in particular to a bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, and a preparation method and application thereof.
Background
In recent years, bioactive peptides have become a word of great energy in the ear. Because of its many potential biological functions, it attracts more and more attention and becomes one of the hot spots of scientific research. The beneficial effects of many bioactive peptides, such as anti-cancer, blood pressure lowering, antibacterial, cholesterol lowering, anti-diabetic, etc., are well documented. Currently more than 3000 different bioactive peptides have been reported in the most authoritative bioactive peptide database BIOPEP-UMW.
Currently, studies on bioactive peptides are mostly focused on food-derived polypeptides, and studies and reports on non-food-derived polypeptides are less. And it has been confirmed from the research results that non-food-derived bioactive peptides have higher affinity and can effectively exert their bioactive functions, compared to food-derived bioactive peptides.
Immunomodulatory peptides are a class of biologically active polypeptides that are first obtained from milk following opioid peptide discovery and demonstrate their physiological activity. Jolles et al found in 1981 for the first time that a hexapeptide with an amino acid sequence Val-Glu-Pro-Ile-Pro-Tyr can be obtained by hydrolyzing human milk protein with trypsin, and in vitro experiments prove that the hexapeptide can enhance the phagocytosis of mouse abdominal cavity macrophages to sheep erythrocytes. Migliore-Samour et al found that the casein-derived hexapeptide Thr-Thr-Met-Pro-Leu-Trp was able to stimulate phagocytosis of murine peritoneal macrophages by sheep red blood cells and to enhance resistance to Klebsiella pneumoniae, with anti-inflammatory properties. Lemna hexandra et al, fed rats with synthetic mouse bone marrow macrophages and a source peptide (PGPIPN), found that phagocytosis of rat peritoneal macrophages and red blood cell-related anti-inflammatory function were significantly enhanced. Bowdis et al, in studying the immune function of the 13 amino acid peptide indolicidin derived from bovine neutrophils, found that the polypeptide indolicidin inhibits LPS-induced TNF- α production in a macrophage-like cell line.
Research shows that the immunoregulation peptide can enhance the immunity of the organism, stimulate the proliferation of lymphocytes of the organism, enhance the phagocytic function of macrophages, promote the release of cytokines, promote the increase of the induced amount of nitric oxide of the macrophages, improve the capability of the organism for resisting the infection of external pathogens, reduce the morbidity of the organism and cannot cause the immune rejection reaction of the organism.
The immunomodulatory peptides presently disclosed are generally small peptides with specific immunomodulatory activity, isolated enzymatically from proteins or synthesized chemically. However, when these small peptides are not enzymatically separated from the protein, the protein itself often has no immunomodulatory activity. It is one of the directions in the field of protein research to find bioactive peptides with specific functions from a wide variety of proteins whose amino acid sequences are known, and to study the functions of these polypeptides.
The amino acid sequence of the Cytochrome C oxidase subunit 7C and mitondrial protein is shown as SEQ ID NO: 2, respectively. At present, no research on related functions of the polypeptide fragment of the Cytochrome C oxidase subunit 7C, mitochondrial protein exists in the prior art.
Disclosure of Invention
The invention aims to provide a bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, and a preparation method and application thereof.
The purpose of the invention can be realized by the following technical scheme:
in a first aspect of the invention, a biologically active polypeptide YFGSGFAAPFFIVRHQLLKK is provided, having an amino acid sequence of Tyr-Phe-Gly-Ser-Gly-Phe-Ala-Ala-Pro-Phe-Phe-Ile-Val-Arg-His-Gln-Leu-Leu-Lys-Lys, as set forth in SEQ ID NO: 1 is shown.
Preferably, the bioactive polypeptide is mouse spleen-derived lymphocyte peptide. The protein is specifically derived from Cytochrome C oxidase subunit 7C and mitochondrial protein, and is an amino acid residue at the 44 th-63 th position of the Cytochrome C oxidase subunit 7C and mitochondrial protein. The amino acid sequence of the Cytochrome C oxidase subunit 7C and mitondrial protein is shown as SEQ ID NO: 2, respectively.
The amino acid sequences and the corresponding nucleotide sequences of the Cytochrome C oxidase subunit 7C and the mitochondrial protein are the prior art, and the nucleotide fragment which codes the Cytochrome C oxidase subunit 7C and the 44 th to 63 th amino acid residues of the mitochondrial protein can code mature bioactive polypeptide YFGSGFAAPFFIVRHQLLKK.
Preferably, the bioactive polypeptide has an anti-inflammatory function and an immunoregulatory function.
The present invention also provides polynucleotides encoding the biologically active peptide YFGSGFAAPFFIVRHQLLKK.
In the second aspect of the present invention, a method for preparing the bioactive polypeptide YFGSGFAAPFFIVRHQLLKK is provided, which can be artificially synthesized by genetic engineering methods, can be directly obtained from cells by a separation and purification method, and can be directly prepared by chemical synthesis.
The artificial synthesis of the bioactive peptide YFGSGFAAPFFIVRHQLLKK by genetic engineering is a technical solution that can be realized by those skilled in the art, and for example, the synthesis of the sequence of the polypeptide can be controlled by a suitable DNA template based on DNA recombination technology.
The method for directly obtaining the cell by the separation and purification method can be as follows: based on the amino acid sequence of the given bioactive peptide YFGSGFAAPFFIVRHQLLKK, the bioactive peptide YFGSGFAAPFFIVRHQLLKK is obtained from mouse spleen-derived lymphocytes by a conventional enzymolysis and purification method in biological technology.
In a third aspect of the invention, the application of the bioactive polypeptide YFGSGFAAPFFIVRHQLLKK in preparing medicines or cosmetics with anti-inflammatory function is provided.
In a fourth aspect of the present invention, there is provided a use of the bioactive peptide YFGSGFAAPFFIVRHQLLKK in the preparation of food or medicine with immunoregulatory function.
Further, the use of the biologically active peptide YFGSGFAAPFFIVRHQLLKK in the manufacture of a food or medicament for promoting an increase in nitric oxide-inducing amount of macrophages.
Further, the use of the bioactive peptide YFGSGFAAPFFIVRHQLLKK in the preparation of a food or medicament for promoting lymphocyte proliferation.
In a fifth aspect of the invention, there is provided an anti-inflammatory product comprising said biologically active polypeptide YFGSGFAAPFFIVRHQLLKK or a derivative of said biologically active polypeptide YFGSGFAAPFFIVRHQLLKK; the anti-inflammatory product comprises anti-inflammatory food, anti-inflammatory health product, anti-inflammatory drug or anti-inflammatory cosmetic.
In a sixth aspect of the present invention, there is provided a product having an immunoregulatory function, comprising said biologically active peptide YFGSGFAAPFFIVRHQLLKK or a derivative of said biologically active peptide YFGSGFAAPFFIVRHQLLKK; the product with immunoregulatory function comprises food with immunoregulatory function or medicine with immunoregulatory function.
Derivatives of the bioactive peptides YFGSGFAAPFFIVRHQLLKK are meant to have the same activity or better activity than the bioactive peptides YFGSGFAAPFFIVRHQLLKK.
The derivative of the biologically active polypeptide YFGSGFAAPFFIVRHQLLKK refers to a polypeptide derivative obtained by performing modifications such as hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation on an amino acid side chain group, an amino terminal or a carboxyl terminal of the biologically active polypeptide YFGSGFAAPFFIVRHQLLKK.
The bioactive polypeptide YFGSGFAAPFFIVRHQLLKK has the following beneficial effects: the bioactive polypeptide YFGSGFAAPFFIVRHQLLKK has good anti-inflammatory activity; the bioactive peptide YFGSGFAAPFFIVRHQLLKK can promote the increase of the induction quantity of macrophage nitric oxide, improve the capability of an organism for resisting the infection of external pathogens, reduce the morbidity of the organism, obviously promote the proliferation of mouse lymphocytes, improve the quality of life and have very important significance for developing foods, health-care products and medicines with the immunoregulation function.
Drawings
FIG. 1: a first order mass spectrum of a fragment with a mass to charge ratio of 582.3296 (m/z 582.3296);
FIG. 2: a secondary mass spectrum of a fragment with a mass-to-charge ratio of 582.3296 and the breakage conditions of the polypeptides az and by;
Detailed Description
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1 Artificial Synthesis of active peptide YFGSGFAAPFFIVRHQLLKK
Synthesis of bioactive peptide
1. 3g of RINK resin (degree of substitution 0.3mmol/g) was weighed into a 150ml reactor and soaked with 50ml of Dichloromethane (DCM).
After 2.2 hours, the resin was washed with 3 resin volumes of N-Dimethylformamide (DMF) and then drained, and this was repeated four times and the resin was drained until use.
3. The Fmoc protecting group on the resin was removed by adding a quantity of 20% piperidine (piperidine/DMF: 1:4, v: v) to the reactor and shaking on a decolourising shaker for 20 min. After deprotection, the resin was washed four times with 3 resin volumes of DMF and then drained.
4. And (3) detecting a small amount of resin by a ninhydrin (ninhydrin) method (detecting A and B, respectively, and reacting at 100 ℃ for 1min), wherein the resin is colored, which indicates that the deprotection is successful.
5. Weighing a proper amount of amino acid Tyr and a proper amount of 1-hydroxy-benzotriazole (HOBT) into a 50ml centrifuge tube, adding 20ml of DMF to dissolve the amino acid Tyr and the 1-hydroxy-benzotriazole (HOBT), then adding 3ml of N, N Diisopropylcarbodiimide (DIC) to shake and shake for 1min, adding the solution into a reactor after the solution is clarified, and then placing the reactor into a 30 ℃ shaking table to react.
After 6.2 hours, the column was capped with a suitable amount of acetic anhydride (acetic anhydride: DIEA: DCM ═ 1:1:2, v: v: v) for half an hour, then washed four times with 3 resin volumes of DMF and drained until needed.
7. The Fmoc protecting group on the resin was removed by adding a quantity of 20% piperidine (piperidine/DMF: 1:4, v: v) to the reactor and shaking on a decolourising shaker for 20 min. After deprotection was washed four times with DMF and then drained.
8. And (3) detecting a small amount of resin by a ninhydrin (ninhydrin) method (detecting A and B, respectively, and reacting at 100 ℃ for 1min), wherein the resin is colored, which indicates that the deprotection is successful.
9. Weighing a second proper amount of amino acid and a proper amount of HOBT in a 50ml centrifuge tube, adding 25ml of DMF to dissolve the amino acid and the HOBT, adding 2.5ml of DIC to shake and shake for 1min, adding the solution into a reactor after the solution is clarified, and then placing the reactor in a shaking table at 30 ℃ to react.
After 10.1 hours, a small amount of resin is taken for detection, and the detection is carried out by an indanthrone method (two drops are respectively detected A and B, and the reaction is carried out for 1min at 100 ℃), if the resin is colorless, the reaction is complete; if the resin is colored, the condensation is not complete and the reaction is continued.
11. After the reaction was completed, the resin was washed four times with DMF and then drained, and a certain amount of 20% piperidine (piperidine/DMF ═ 1:4, v: v) was added to the reactor, and the mixture was shaken on a decolorizing shaker for 20min to remove the Fmoc-protecting group from the resin. After the protection is removed, washing with DMF for four times, and then draining to detect whether the protection is removed.
12. The amino acids Tyr, Phe, Gly, Ser, Gly, Phe, Ala, Pro, Phe, Ile, Val, Arg, His, Gln, Leu, Lys are grafted in sequence according to steps 9-11.
13. After the last amino acid had been grafted, the protection was removed, washed four times with DMF and the resin was drained with methanol. The polypeptide was then cleaved from the resin with 95 cleavage medium (trifluoroacetic acid: 1,2 ethanedithiol: 3, isopropylsilane: water: 95:2:2:1, v: v: v) (10 ml of cleavage medium per gram of resin) and centrifuged four times with glacial ethyl ether (cleavage medium: ethyl ether: 1:9, v: v).
To this end, bioactive peptide YFGSGFAAPFFIVRHQLLKK was synthesized.
Confirmation of biologically active peptides
1) UPLC analysis
UPLC conditions were as follows:
the instrument comprises the following steps: waters ACQUITY UPLC ultra-high performance liquid phase, electrospray, quadrupole and time-of-flight mass spectrometer
Specification of chromatographic column: BEH C18 chromatographic column
Flow rate: 0.4mL/min
Temperature: 50 deg.C
Ultraviolet detection wavelength: 210nm
Sample introduction amount: 2 μ L
Gradient conditions: solution A: water containing 0.1% formic acid (v/v), liquid B: acetonitrile containing 0.1% formic acid (v/v)
2) Mass spectrometric analysis
The mass spectrometry conditions were as follows:
ion mode: ES +
Mass range (m/z): 100. 1000A
Capillary voltage (Capillary) (kV): 3.0
Sampling cone (V): 35.0
Ion source temperature (. degree. C.): 115
Desolvation temperature (. degree. C.): 350
Desolventizing gas stream (L/hr): 700.0
Collision energy (eV): 4.0
Scan time (sec): 0.25
Inner scan time (sec): 0.02
According to the above analysis method, the bioactive peptide YFGSGFAAPFFIVRHQLLKK was subjected to chromatographic analysis and mass spectrometric analysis using ultra high performance liquid, electrospray, quadrupole, time-of-flight mass spectrometry. The primary mass spectrum of the bioactive peptide YFGSGFAAPFFIVRHQLLKK is shown in figure 1, the secondary mass spectrum of the extracted peak and the az and by breaking conditions are shown in figure 2, the polypeptide mass-to-charge ratio of the peak is 582.3296, and the retention time is 51.58 min.
3) Results
As can be seen from fig. 2, the fragment sequence of mass-to-charge ratio 582.3296 was determined by Mascot software analysis and calculation based on az and by cleavage, to be Tyr, Phe, Gly, Ser, Gly, Phe, Ala, Pro, Phe, Ile, Val, Arg, His, Gln, Leu, Lys (YFGSGFAAPFFIVRHQLLKK), and was identified as SEQ ID NO: 1. the fragment corresponds to a residue sequence of 44-63 sites of Cytochrome C oxidase subunit 7C and mitochondrial protein, the GenBank number of the amino acid sequence of Cytochrome C oxidase subunit 7C and mitochondrial protein is BAE20843.1, and the sequence is shown in SEQ ID NO: 2.
example 2 immunological Activity assay of bioactive peptides
In vitro lymphocyte proliferation capacity experiment (MTT method) of bioactive polypeptide YFGSGFAAPFFIVRHQLLKK
1. Experimental materials and instruments:
reagents and materials: experimental animals balb/c mice (male 6-8 weeks old, animal experiment center of Shanghai university of transportation, college of agriculture and biology); the mouse spleen lymphocyte-derived bioactive peptide YFGSGFAAPFFIVRHQLLKK obtained in example 1; mouse lymphocyte extract (ex solibao); RPMI1640 medium (purchased from GIBCO); 3- (4, 5-Dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide salt (MTT, available from Amresco, Inc.); concanavalin (ConA, available from Sigma); bovine serum albumin (BSA, available from Genebase); pepsin (available from Sigma); pancreatin (Corolase PP, from AB).
The instrument equipment comprises: LRH-250F Biochemical incubator, Shanghai Hengshi Co., Ltd; GL-22M high speed refrigerated centrifuge, shanghai luxiang instrument centrifuge instruments ltd; hera cell 150CO2Incubator, Heraeus corporation; dragon Wellscan MK3 microplate reader, Labsystems Inc.; ALPHA 1-2-LD vacuum freeze drier, Christ company; ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometer, waters corporation.
2. The experimental method comprises the following steps:
taking mouse spleen under aseptic condition, extracting mouse lymphocyte with lymphocyte extract, and performing primary culture. The cell density was adjusted to 2.5X 10 with complete RPMI1640 medium6one/mL. To a 96-well cell culture plate were added in sequence: 100 μ L mouse lymphocyte suspension, 100 μ L RPMI1640 complete medium, 20 μ L concanavalin, 100 μ L polypeptide sample. In addition, a blank control group (PBS with pH7.2-7.4 and 3 mol/L) and a negative control group (500 mu g/mL BSA) are arranged, and the research shows that the blank control group has no influence on the in vitro lymphocyte proliferation. Each set of 3 replicates. At 5% CO2Culturing at 37 deg.C for 68h, adding 20 μ L MTT into each well under aseptic condition, culturing for 4h, carefully removing supernatant, adding 100 μ L dimethyl sulfoxide into each well, incubating at 37 deg.C for 10min, shaking, and measuring absorbance at 570nm with microplate reader.
The in vitro lymphocyte proliferation capacity is expressed by a stimulation index and is calculated as follows:
in the formula: a. the1Absorbance at 570nm for the blank; a. the2Absorbance at 570nm for the negative control, A3The absorbance at 570nm for the experimental group.
3. Experimental results and analysis:
TABLE 2 Effect of the bioactive polypeptide YFGSGFAAPFFIVRHQLLKK on in vitro lymphocyte proliferation
Experiment grouping | Stimulation index SI |
BSA | 1 |
YFGSGFAAPFFIVRHQLLKK | 1.482±0.021** |
Note: the number marked as significant difference (P < 0.05) compared to the negative control.
The difference in the negative control group was very significant (P <0.01)
The results are shown in Table 2. As shown in Table 2, under the condition that the mass concentration of the bioactive peptide YFGSGFAAPFFIVRHQLLKK is 200. mu.g/mL, the stimulation index of the bioactive peptide YFGSGFAAPFFIVRHQLLKK is greater than that of BSA, which indicates that YFGSGFAAPFFIVRHQLLKK can stimulate the proliferation of mouse lymphocytes in vitro to a certain extent. And YFGSGFAAPFFIVRHQLLKK reached a stimulation index of 1.482, which was very significantly different from the negative control group (P < 0.01). Therefore, the active polypeptide YFGSGFAAPFFIVRHQLLKK can be considered to have the capacity of remarkably promoting the mouse lymphocyte proliferation, can be used as a substance with immunoregulation activity to be added into health products, and can improve the immunity of human bodies.
Second, determination of macrophage-promoting nitric oxide-inducing amount of bioactive polypeptide YFGSGFAAPFFIVRHQLLKK (Griess method)
1. Experimental reagents and instruments:
reagent: experimental animal balb/c mouse (male 6-8 weeks old) spleen lymphocyte source bioactive peptide YFGSGFAAPFFIVRHQLLKK; LPS, purchased from Sigma; neutral red staining solution, produced by Biyuntian biotechnological research institute.
The instrument equipment comprises: LRH-250F Biochemical incubator Shanghai Hengshi Co., Ltd; GL-22M high speed refrigerated centrifuge Shanghai Luxiang apparatus centrifuge Instrument Co., Ltd; hera cell 150CO2Incubator Heraeus; dragon Wellscan MK3 microplate reader Labsystems.
2. The test method comprises the following steps:
the number of the added cells was 2X 106100 μ l/well of a cell suspension per ml, 200 μ l/well of a complete peptide-containing RPMI1640 culture medium (10% FBS) was added after adherent purification, LPS was added to a final concentration of 10 μ g/ml at 24 hours in an inflammation group, 50 μ l/well of a culture supernatant was collected after continuous culture for 48 hours, 50 μ l/well of Griess reagent 1 and Griess reagent 2 were sequentially added to the culture supernatant, and after reaction at room temperature for 10 minutes, an absorbance value (OD540) was measured at a wavelength of 540 nm.
3. Experimental results and analysis:
TABLE 1 determination of macrophage nitric oxide-inducing amount of biologically active polypeptide YFGSGFAAPFFIVRHQLLKK
Experiment grouping | Normal group | Inflammation group |
Cell blank | 0.0783±0.649 | 0.3389±0.0427 |
Polypeptide (1mg/ml) | 0.765±0.0422 | 0.4573±0.0321** |
Polypeptide (0.5mg/ml) | 0.1472±0.0248** | 0.4837±0.0129** |
Note: significant difference compared to negative control (P < 0.05);
the difference in the negative control group was very significant (P <0.01)
The results are shown in table 1, and it is understood from table 1 that the addition of biologically active polypeptide YFGSGFAAPFFIVRHQLLKK at a concentration of 0.5mg/mL to the experimental group promoted the nitric oxide-induced amount of macrophages both under normal conditions and under conditions of LPS-induced inflammation, and showed a significant difference (P <0.01) compared to the cell blank group. When the added concentration of the bioactive polypeptide YFGSGFAAPFFIVRHQLLKK is 1mg/mL, the increase of the nitric oxide induction amount of macrophages can be promoted under the condition of inflammation caused by LPS, and the difference is very significant (P < 0.01). But there were no significant differences compared to the cell blank grown under normal conditions. The biologically active polypeptide YFGSGFAAPFFIVRHQLLKK is shown to have the ability to promote an increase in nitric oxide-induced levels in macrophages at certain concentrations.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Sequence listing
<110> panda milk group GmbH, Zhejiang ghui peptide Life health science & technology, Inc
<120> a bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, and its preparation method and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Tyr Phe Gly Ser Gly Phe Ala Ala Pro Phe Phe Ile Val Arg His Gln
1 5 10 15
Leu Leu Lys Lys
20
<210> 2
<211> 63
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Leu Gly Gln Ser Ile Arg Arg Phe Thr Thr Ser Val Val Arg Arg
1 5 10 15
Ser His Tyr Glu Glu Gly Pro Gly Lys Asn Leu Pro Phe Ser Val Glu
20 25 30
Asn Lys Trp Arg Leu Leu Ala Met Met Thr Val Tyr Phe Gly Ser Gly
35 40 45
Phe Ala Ala Pro Phe Phe Ile Val Arg His Gln Leu Leu Lys Lys
50 55 60
Claims (10)
1. A bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, wherein the amino acid is Tyr-Phe-Gly-Ser-Gly-Phe-Ala-Ala-Pro-Phe-Phe-Ile-Val-Arg-His-Gln-Leu-Leu-Lys-Lys.
2. A polynucleotide encoding the biologically active peptide YFGSGFAAPFFIVRHQLLKK of claim 1.
3. The method of claim 1, wherein the biologically active polypeptide YFGSGFAAPFFIVRHQLLKK is synthesized by genetic engineering, obtained directly from cells by isolation and purification, or prepared directly by chemical synthesis.
4. The use of biologically active polypeptide YFGSGFAAPFFIVRHQLLKK of claim 1, wherein the biologically active polypeptide YFGSGFAAPFFIVRHQLLKK is used in the manufacture of a medicament or cosmetic product with anti-inflammatory properties.
5. The use of biologically active peptide YFGSGFAAPFFIVRHQLLKK of claim 1, wherein the use of biologically active peptide YFGSGFAAPFFIVRHQLLKK in the preparation of a food or a medicament having immunomodulatory properties.
6. The use of biologically active peptide YFGSGFAAPFFIVRHQLLKK of claim 5 wherein said biologically active peptide YFGSGFAAPFFIVRHQLLKK is used in the manufacture of a food or medicament for promoting increased nitric oxide-induced production of macrophages.
7. The use of biologically active peptide YFGSGFAAPFFIVRHQLLKK of claim 5, wherein said biologically active peptide YFGSGFAAPFFIVRHQLLKK is used in the manufacture of a food or a medicament for promoting lymphocyte proliferation.
8. An anti-inflammatory product comprising the biologically active polypeptide YFGSGFAAPFFIVRHQLLKK of claim 1 or a derivative of the biologically active polypeptide YFGSGFAAPFFIVRHQLLKK; the anti-inflammatory product comprises an anti-inflammatory drug or an anti-inflammatory cosmetic; derivatives of the bioactive peptides YFGSGFAAPFFIVRHQLLKK are meant to have the same activity or better activity than the bioactive peptides YFGSGFAAPFFIVRHQLLKK.
9. A product having an immunomodulatory function, comprising the biologically active peptide YFGSGFAAPFFIVRHQLLKK of claim 1 or a derivative of the biologically active peptide YFGSGFAAPFFIVRHQLLKK; the product with immunoregulation function comprises food with immunoregulation function or medicine with immunoregulation function; derivatives of the bioactive peptides YFGSGFAAPFFIVRHQLLKK are meant to have the same activity or better activity than the bioactive peptides YFGSGFAAPFFIVRHQLLKK.
10. An anti-inflammatory product according to claim 8 or an immunomodulatory product according to claim 9, wherein the derivative of the biologically active polypeptide YFGSGFAAPFFIVRHQLLKK is a polypeptide derivative obtained by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation modification at the amino acid side chain group, amino terminus or carboxy terminus of the biologically active polypeptide YFGSGFAAPFFIVRHQLLKK.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011485267.6A CN112501140B (en) | 2020-12-16 | 2020-12-16 | Bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, and preparation method and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011485267.6A CN112501140B (en) | 2020-12-16 | 2020-12-16 | Bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, and preparation method and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112501140A true CN112501140A (en) | 2021-03-16 |
CN112501140B CN112501140B (en) | 2022-03-18 |
Family
ID=74972489
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011485267.6A Active CN112501140B (en) | 2020-12-16 | 2020-12-16 | Bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, and preparation method and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112501140B (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112574291A (en) * | 2020-12-14 | 2021-03-30 | 上海交通大学 | Bioactive peptide SHRKFSAPRHGSLGFLPR, and preparation method and application thereof |
CN112592396A (en) * | 2020-12-14 | 2021-04-02 | 上海交通大学 | Bioactive peptide VDPFSKKDW, and preparation method and application thereof |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104535765A (en) * | 2009-03-12 | 2015-04-22 | 癌症预防和治疗有限公司 | Methods of identification, assessment, prevention and therapy of lung diseases and kits thereof |
CN107163136A (en) * | 2017-07-06 | 2017-09-15 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide WNIPMGLIVNQ and its preparation method and application |
CN107200782A (en) * | 2017-07-06 | 2017-09-26 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide VAVVKKGSNFQ and its preparation method and application |
CN107815443A (en) * | 2016-09-14 | 2018-03-20 | 清华大学 | A kind of super complex proteins of respiratory chain |
US10293058B2 (en) * | 2015-04-22 | 2019-05-21 | Curevac Ag | RNA containing composition for treatment of tumor diseases |
CN112724237A (en) * | 2021-01-19 | 2021-04-30 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide GGSDGYGSGRGF, and preparation method and application thereof |
-
2020
- 2020-12-16 CN CN202011485267.6A patent/CN112501140B/en active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104535765A (en) * | 2009-03-12 | 2015-04-22 | 癌症预防和治疗有限公司 | Methods of identification, assessment, prevention and therapy of lung diseases and kits thereof |
US10293058B2 (en) * | 2015-04-22 | 2019-05-21 | Curevac Ag | RNA containing composition for treatment of tumor diseases |
CN107815443A (en) * | 2016-09-14 | 2018-03-20 | 清华大学 | A kind of super complex proteins of respiratory chain |
CN107163136A (en) * | 2017-07-06 | 2017-09-15 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide WNIPMGLIVNQ and its preparation method and application |
CN107200782A (en) * | 2017-07-06 | 2017-09-26 | 浙江辉肽生命健康科技有限公司 | A kind of biologically active polypeptide VAVVKKGSNFQ and its preparation method and application |
CN112724237A (en) * | 2021-01-19 | 2021-04-30 | 浙江辉肽生命健康科技有限公司 | Bioactive peptide GGSDGYGSGRGF, and preparation method and application thereof |
Non-Patent Citations (8)
Title |
---|
GENBANK: "Chain l, Cytochrome c oxidase subunit 7C, mitochondrial", 《GENBANK》 * |
GENBANK: "mCG1735, isoform CRA_b [Mus musculus]", 《GENBANK》 * |
R S SEELAN 等: "Structural organization and promoter analysis of the bovine cytochrome c oxidase subunit VIIc gene. A functional role for YY1", 《J BIOL CHEM》 * |
UNIPROTKB: "UniProtKB-B8JJA9 (B8JJA9_MOUSE)", 《UNIPROTKB》 * |
WWW.CELL.COM: "mmc2,https://www.cell.com/cms/10.1016/j.cell.2011.10.002/attachment/a547d5ad-5935-4607-bd0b-ed5a89623b97/mmc2", 《WWW.CELL.COM》 * |
梁宋平: "《生物化学与分子生物学实验教程》", 31 March 2003, 高等教育出版社 * |
梁清华 等: "黄芩苷对T淋巴细胞增殖与活化的影响", 《全国第八届中西医结合风湿病学术会议论文汇编》 * |
陈刚 等: "白芍总苷对巨噬细胞一氧化氮和诱导型一氧化氮合酶生成的影响及其机制研究", 《中国免疫学杂志》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112574291A (en) * | 2020-12-14 | 2021-03-30 | 上海交通大学 | Bioactive peptide SHRKFSAPRHGSLGFLPR, and preparation method and application thereof |
CN112592396A (en) * | 2020-12-14 | 2021-04-02 | 上海交通大学 | Bioactive peptide VDPFSKKDW, and preparation method and application thereof |
CN112592396B (en) * | 2020-12-14 | 2022-10-21 | 上海交通大学 | Bioactive peptide VDPFSKKDW as well as preparation method and application thereof |
Also Published As
Publication number | Publication date |
---|---|
CN112501140B (en) | 2022-03-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112759636B (en) | Bioactive peptide with amino acid structure ESLKGVDPKFLR, and preparation method and application thereof | |
CN112501140B (en) | Bioactive polypeptide YFGSGFAAPFFIVRHQLLKK, and preparation method and application thereof | |
CN112812168A (en) | Bioactive peptide GLNMCRQCF, and preparation method and application thereof | |
CN112625113A (en) | Bioactive peptide AGYDVEKNNSRIKLGLK, and preparation method and application thereof | |
CN112646022A (en) | Bioactive peptide PKCPKCDKEVYFAERV, and preparation method and application thereof | |
CN112661830B (en) | Bioactive peptide with amino acid structure AIRNDEELNKLLGR, and preparation method and application thereof | |
CN112724237B (en) | Bioactive peptide GGSDGYGSGRGF, and preparation method and application thereof | |
CN112500469B (en) | Bioactive polypeptide AAPAAPAAAPPAE, and preparation method and application thereof | |
CN112480233B (en) | Bioactive peptide IAHPKLGKRIR, and preparation method and application thereof | |
CN112480232A (en) | Bioactive peptide VSLADLQNDEVAFR, and preparation method and application thereof | |
CN112500468A (en) | Bioactive peptide RLAFIAHPKLG, and preparation method and application thereof | |
CN112745379A (en) | Bioactive peptide with amino acid structure RDNKKTRIIPR, and preparation method and application thereof | |
CN112500467A (en) | Bioactive peptide RRECPSDECGAGVF, and preparation method and application thereof | |
CN112480234A (en) | Bioactive peptide AAGGYDVEKNNSRIKLGLK, and preparation method and application thereof | |
CN112724239B (en) | Bioactive peptide with amino acid structure NKELDPVQKLFVDKIREYK and application thereof | |
CN112592396A (en) | Bioactive peptide VDPFSKKDW, and preparation method and application thereof | |
CN112759634B (en) | Bioactive peptide with amino acid structure FEYIEENKY, and preparation method and application thereof | |
CN112812170B (en) | Bioactive polypeptide with amino acid structure LLPKKTE as well as preparation method and application thereof | |
CN112778410B (en) | Bioactive peptide SAPRHGSLGFLPRK, and preparation method and application thereof | |
CN112745382B (en) | Bioactive peptide with amino acid structure LTVINQTQKENLR, and preparation method and application thereof | |
CN112724238B (en) | Bioactive peptide with amino acid structure FREGTTPKPK, and preparation method and application thereof | |
CN112646023B (en) | Bioactive peptide with amino acid structure VNVVPTFGKKKGP, and preparation method and application thereof | |
CN112812171B (en) | Bioactive peptide with amino acid structure VVRKPLNKEGKKP, and preparation method and application thereof | |
CN112481246B (en) | Bioactive polypeptide FSPANKKLTPKKY, and preparation method and application thereof | |
CN112812169B (en) | Bioactive peptide with amino acid structure APKIQRLVTPR, and preparation method and application thereof |
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 |