CN113801198B - 一种杂合抗菌肽及其制备方法和应用 - Google Patents
一种杂合抗菌肽及其制备方法和应用 Download PDFInfo
- Publication number
- CN113801198B CN113801198B CN202110893482.8A CN202110893482A CN113801198B CN 113801198 B CN113801198 B CN 113801198B CN 202110893482 A CN202110893482 A CN 202110893482A CN 113801198 B CN113801198 B CN 113801198B
- Authority
- CN
- China
- Prior art keywords
- peptide
- antibacterial
- hybrid
- antibacterial peptide
- additive
- 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
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 67
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- 241000588724 Escherichia coli Species 0.000 claims abstract description 5
- 241000607142 Salmonella Species 0.000 claims abstract description 4
- 108020004707 nucleic acids Proteins 0.000 claims description 16
- 102000039446 nucleic acids Human genes 0.000 claims description 16
- 150000007523 nucleic acids Chemical class 0.000 claims description 16
- 239000000654 additive Substances 0.000 claims description 15
- 230000000996 additive effect Effects 0.000 claims description 15
- 239000013604 expression vector Substances 0.000 claims description 15
- 108700042778 Antimicrobial Peptides Proteins 0.000 claims description 13
- 102000044503 Antimicrobial Peptides Human genes 0.000 claims description 13
- 239000003814 drug Substances 0.000 claims description 6
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims description 5
- 239000003674 animal food additive Substances 0.000 claims description 5
- 239000000490 cosmetic additive Substances 0.000 claims description 3
- 235000013373 food additive Nutrition 0.000 claims description 3
- 239000002778 food additive Substances 0.000 claims description 3
- 239000003112 inhibitor Substances 0.000 claims description 3
- 230000000813 microbial effect Effects 0.000 claims description 3
- 239000003755 preservative agent Substances 0.000 claims description 3
- 230000002335 preservative effect Effects 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims description 2
- 239000000825 pharmaceutical preparation Substances 0.000 claims 1
- 229940127557 pharmaceutical product Drugs 0.000 claims 1
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 33
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 30
- 241000894006 Bacteria Species 0.000 abstract description 9
- 108010091867 peptide P Proteins 0.000 abstract description 8
- 239000003242 anti bacterial agent Substances 0.000 abstract description 5
- 241000192125 Firmicutes Species 0.000 abstract description 4
- 244000052616 bacterial pathogen Species 0.000 abstract description 4
- 235000019730 animal feed additive Nutrition 0.000 abstract description 3
- 230000006806 disease prevention Effects 0.000 abstract description 3
- 238000009920 food preservation Methods 0.000 abstract description 3
- 239000002253 acid Substances 0.000 abstract description 2
- 238000005453 pelletization Methods 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 19
- 230000002354 daily effect Effects 0.000 description 18
- 206010012735 Diarrhoea Diseases 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 102000004196 processed proteins & peptides Human genes 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 7
- 241000282887 Suidae Species 0.000 description 7
- 235000021050 feed intake Nutrition 0.000 description 7
- 230000006872 improvement Effects 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 101710116112 Protonectin Proteins 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 229940079593 drug Drugs 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 108010048087 melectin Proteins 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- KSVPSFMHPLYOBT-VDAJQBIKSA-N (2s)-1-[(2s)-2-[[(2s)-2-[[(2s)-6-amino-2-[[(2s)-6-amino-2-[[(2s)-2-[[(2s,3s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[(2-aminoacetyl)amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxypropanoyl]amino]-3-methylpentanoyl]amino]-4-methylpentanoyl]amino]h Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(N)=O)NC(=O)CN)C1=CC=CC=C1 KSVPSFMHPLYOBT-VDAJQBIKSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 230000002949 hemolytic effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000003359 percent control normalization Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 2
- 238000008157 ELISA kit Methods 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- SEQKRHFRPICQDD-UHFFFAOYSA-N N-tris(hydroxymethyl)methylglycine Chemical compound OCC(CO)(CO)[NH2+]CC([O-])=O SEQKRHFRPICQDD-UHFFFAOYSA-N 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 101710177166 Phosphoprotein Proteins 0.000 description 2
- 241001138501 Salmonella enterica Species 0.000 description 2
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 239000002435 venom Substances 0.000 description 2
- 210000001048 venom Anatomy 0.000 description 2
- 231100000611 venom Toxicity 0.000 description 2
- 239000000273 veterinary drug Substances 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- RBCOYOYDYNXAFA-UHFFFAOYSA-L (5-hydroxy-4,6-dimethylpyridin-3-yl)methyl phosphate Chemical compound CC1=NC=C(COP([O-])([O-])=O)C(C)=C1O RBCOYOYDYNXAFA-UHFFFAOYSA-L 0.000 description 1
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 241001255879 Agelaia pallipes Species 0.000 description 1
- 108010050820 Antimicrobial Cationic Peptides Proteins 0.000 description 1
- 102000014133 Antimicrobial Cationic Peptides Human genes 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000212959 Cryptotaenia Species 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241001360526 Escherichia coli ATCC 25922 Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- YYOCMTFVGKDNQP-IHRRRGAJSA-N His-Met-Lys Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N YYOCMTFVGKDNQP-IHRRRGAJSA-N 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- MJVAVZPDRWSRRC-UHFFFAOYSA-N Menadione Chemical compound C1=CC=C2C(=O)C(C)=CC(=O)C2=C1 MJVAVZPDRWSRRC-UHFFFAOYSA-N 0.000 description 1
- IMTUWVJPCQPJEE-IUCAKERBSA-N Met-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN IMTUWVJPCQPJEE-IUCAKERBSA-N 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- UZMAPBJVXOGOFT-UHFFFAOYSA-N Syringetin Natural products COC1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UZMAPBJVXOGOFT-UHFFFAOYSA-N 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 239000007997 Tricine buffer Substances 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
- 108010084455 Zeocin Proteins 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 230000002141 anti-parasite Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 239000003096 antiparasitic agent Substances 0.000 description 1
- 235000021052 average daily weight gain Nutrition 0.000 description 1
- 235000014590 basal diet Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229940054666 biotin 0.15 mg Drugs 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000002815 broth microdilution Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- FDJOLVPMNUYSCM-UVKKECPRSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2,7, Chemical compound [Co+3].N#[C-].C1([C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)[N-]\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O FDJOLVPMNUYSCM-UVKKECPRSA-L 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- KCFYHBSOLOXZIF-UHFFFAOYSA-N dihydrochrysin Natural products COC1=C(O)C(OC)=CC(C2OC3=CC(O)=CC(O)=C3C(=O)C2)=C1 KCFYHBSOLOXZIF-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229940094710 folic acid 1.2 mg Drugs 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 229940055726 pantothenic acid Drugs 0.000 description 1
- 235000019161 pantothenic acid Nutrition 0.000 description 1
- 239000011713 pantothenic acid Substances 0.000 description 1
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- -1 polypropylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- 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
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3526—Organic compounds containing nitrogen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
本发明公开了一种杂合抗菌肽及其制备方法和应用,所述杂合抗菌肽的氨基酸序列如SEQ ID NO:1所示。本发明中的杂合抗菌肽Melectin‑P具有广谱抗菌活性,对多数革兰氏阳性菌及革兰氏阴性菌均有不同程度的抑菌活性,特别是对大肠杆菌、沙门氏菌、金黄色葡萄球菌具有较好的抑菌活性。同原始抗菌肽相比,杂合后抗菌肽对常见致病菌的抑菌活性显著增强。抑菌特别是同时又具有热稳定性和酸稳定性的特点,使其在食品防腐、疾病防治和动物饲料添加剂等方面显示很好的应用前景。而且本发明中的杂合肽Melectin‑P是小分子肽,具有热稳定性,可以在100℃的高温保持45min,可以满足饲料制粒的高温要求。
Description
技术领域
本发明属于生物技术领域,具体涉及一种杂合抗菌肽及其制备方法和应用。
背景技术
饲粮中添加抗生素可缓解仔猪断奶应急,促进仔猪生长,但抗生素残留及病原菌耐药性问题对畜禽及人类健康产生了负面的影响。2020年,我们国家畜禽养殖用饲料全面禁抗,饲用抗生素的禁用会造成仔猪保育前期的发病死淘率增加,生长速度降低。在饲料禁抗的背景下,寻求有效的抗生素替代物,提升猪肠道免疫调节作用,研发新型高效安全的产品来改善生猪肠道健康就显得尤其重要。
抗菌肽是一类具有广谱抑菌活性的多肽类物质,部分抗菌肽还能激活免疫系统,在细胞信号传导过程、细胞增殖调节过程以及抗肿瘤、抗病毒、抗寄生虫等方面具有重要功能。抗菌肽因其独特的杀菌机制和优良的理化性质成为抗生素替代品研发的热点,抗菌肽在医用抗感染治疗药物以及畜牧业生产中具有广阔应用前景。在抗菌肽研发过程中,改造已有抗菌肽和设计新抗菌肽分子是提高其活性的有效途径。对抗菌肽进行分子设计、结构改造和修饰、降低其溶血活性、提高其抑菌活性和酸碱稳定性等成为抗菌肽研发的重要内容之一。
Melectin是分离自隐寄生蜂毒液的一种抗菌肽,可抑制革兰氏阳性和阴性菌,具有较低的溶血活性。Protonectin是分离自新大陆热带区社会黄蜂Agelaia pallipes毒液的阳离子型抗菌肽。其对大肠杆菌、绿脓杆菌、枯草芽孢杆菌、金色葡萄球菌都有一定的抑制作用。
目前,改造已有抗菌肽和设计新抗菌肽分子是提高其活性的有效途径。采用基因工程技术生产抗菌肽具有活性高、产量大、成本低廉、生产速度快、易于规模化生产等优点。目前抗菌肽的抗菌活性较抗生素还存在一定的差距,且很多抗菌肽还存在着毒性强、稳定性差、成本高等诸多问题。因此,对抗菌肽进行分子设计、结构改造和修饰、降低其溶血活性、提高、提高其抑菌效果及广泛性、提高其热稳定性、酸碱稳定性等成为抗菌肽研发的重要内容之一。
发明内容
本发明的第一个目的在于提供一种杂合抗菌肽。
本发明的第二个目的在于提供一种核酸分子。
本发明的第三个目的在于提供一种表达载体。
本发明的第四个目的在于提供一种重组细胞。所述细胞非植物或动物细胞。
本发明的第五个目的在于提供上述抗菌肽或核酸分子或表达载体或重组细胞在制备微生物抑制剂中的应用。
本发明的第六个目的在于提供上述抗菌肽或核酸分子或表达载体或重组细胞在制备饲料或添加剂中的应用。
本发明的第七个目的在于提供一种添加剂。
本发明所采取的技术方案是:
本发明的第一个方面,提供一种杂合抗菌肽Melectin-P,其氨基酸序列如SEQ IDNO:1所示。
本发明的第二个方面,提供一种核酸分子,编码本发明第一方面所述抗菌肽。
在本发明的一些优选实施方式中,所述核酸分子的序列如SEQ ID NO:2所示。
本发明的第三个方面,提供一种表达载体,包含本发明第二方面所述核酸分子。
本发明的第四个方面,提供一种重组细胞,包含本发明第三方面所述表达载体。
在本发明的一些实施方式中,所述细胞非植物细胞或动物细胞。
本发明的第五个方面,提供本发明第一方面所述杂合抗菌肽或本发明第二方面所述核酸分子或本发明第三方面所述表达载体或本发明第四方面所述重组细胞在制备微生物抑制剂中的应用。
在本发明的一些实施方式中,所述微生物为大肠杆菌、沙门氏菌、铜绿假单胞菌或金黄色葡萄球菌。
本发明的第六个方面,提供本发明第一方面所述杂合抗菌肽或本发明第二方面所述核酸分子或本发明第三方面所述表达载体或本发明第四方面所述重组细胞在制备饲料或添加剂中的应用。
在本发明的一些优选实施方式中,所述添加剂为防腐剂或药。
在本发明的一些优选实施方式中,所述药为兽药。
在本发明的一些实施方式中,所述添加剂为食品添加剂、饲料添加剂、化妆品添加剂或卫生产品添加剂。
本发明的第七个方面,提供一种添加剂,包含本发明第一方面所述杂合抗菌肽。
在本发明的一些实施方式中,所述添加剂为防腐剂或药。
在本发明的一些优选实施方式中,所述药为兽药。
在本发明的一些实施方式中,所述添加剂为食品添加剂、饲料添加剂、化妆品添加剂或卫生产品添加剂。
在本发明的一些实施方式中,所述饲料添加剂的使用量为0.1~1%。
在本发明的一些优选实施方式中,所述饲料添加剂的使用量为0.2%。
本发明的有益效果是:
本发明提供一种杂合抗菌肽Melectin-P、编码抗菌肽的核酸分子以及包含上述核酸分子的表达载体或重组细胞。本发明中的杂合抗菌肽Melectin-P具有广谱抗菌活性,对多数革兰氏阳性菌及革兰氏阴性菌均有不同程度的抑菌活性,特别是对大肠杆菌、沙门氏菌、金黄色葡萄球菌具有较好的抑菌活性。同原始抗菌肽相比,杂合后抗菌肽对常见致病菌的抑菌活性显著增强。抑菌特别是同时又具有热稳定性和酸稳定性的特点,使其在食品防腐、疾病防治和动物饲料添加剂等方面显示很好的应用前景。而且本发明中的杂合肽Melectin-P是小分子肽,可以在100℃的高温保持45min,具有较好的热稳定性,可以满足饲料制粒的高温要求。
附图说明
图1为重组杂合肽Melectin-P的Tricine-SDS-PAGE电泳检测。其中条带2-7分别为重组菌发酵0h、24h、48h、72h、96h和120h的电泳图;条带1和8为Marker。
图2为重组杂合肽Melectin-P的热稳定性图。
图3为重组杂合肽Melectin-P的酸碱稳定性图。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述,以充分地理解本发明的目的、特征和效果。显然,所描述的实施例只是本发明的一部分实施例,而不是全部实施例,基于本发明的实施例,本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例,均属于本发明保护的范围。
毕赤酵母X-33(His-Mut+)、表达载体pPICZα-A购于Invitrogen公司;Escherichiacoli ATCC25922、Pseudomonas aeruginosa ATCC 19429、Staphylococcus aureusATCC6538、Salmonella enterica ATCC13076、Salmonella typhimurium ATCC14028为本实验室保存。
限制性内切酶XholⅠ、XbalⅠ、SacⅠ、T4连接酶购自Takara(大连)有限公司;Zeocin、Tricine、琼脂糖凝胶回收试剂盒、质粒提取试剂盒、DNA分子质量Marker、小分子质量蛋白Maker购自上海生工生物工程技术服务有限公司;pMD18-T由本实验室保藏;其他试剂为国产分析纯;多核苷酸及DNA测序由华大基因完成。
实施例1抗菌肽的制备
(1)杂合抗菌肽Melectin-P基因的克隆
在Melectin和Protonectin的序列、结构分析的基础上,进行杂合优化,选择Melectin的8-18位氨基酸与Protonectin的3-10位氨基酸进行杂合,获得一种新型的杂合抗菌肽,抗菌肽的具体信息见表1。
表1抗菌肽具体信息
选用酵母偏爱密码子设计Melectin-P杂合肽基因,其基因序列为GGTACTATTTTGGGA CTGTTGAAGAAAGTGCTGCCAAAGGTCATGGCTCACATGAAA(SEQ ID NO:2),其编码的氨基酸序列为GTILGLLKKVLPKVMAHMK(SEQ ID NO:1)。在Melectin-P杂合肽N端加上α信号肽Kex2裂解位点(-Glu-Lys-Arg-)。化学合成具有酵母密码子偏爱性编码基因,并在编码基因5'和3'端分别引入XholⅠ、XbalⅠ酶切位点。应用SOE-PCR的方法(两次PCR)合成所需要的目的基因。
反应体系(25μL):10×PCR Buffer(Mg2+free)2.5μL,MgCl2(25mmol/L)1.5μL,dNTP(10mmol/L)0.5μL,F1、F2(100pmol/L)各0.5μL,TaKaRa Ex TaqTM 0.25μL,灭菌超纯水19.25μL。混匀,瞬间离心。PCR反应条件:94℃预变性2min,进入PCR循环:94℃30s,退火温度从65℃降至50℃,每循环1min,每一个循环降低0.5℃,72℃1min,共30个循环后温度降至50℃,再在最适退火温度55℃的条件下进行15个循环;最后72℃延伸6min。取产物7.5μL,2%琼脂糖凝胶电泳。
(2)重组酵母表达载体的构建和Melectin-P的诱导表达
经SOE合成好的基因经XhoⅠ、XbaⅠ双酶切,定向插入酵母表达载体pPICZα-A中,构建Melectin-P重组质粒,缺失酶切位点鉴定阳性的质粒命名为pPICZα-A-Melectin-P,送深圳华大基因测序。将测序正确的重组质粒转化酵母菌X-33,重组酵母的电击转化、筛选按Invitrogen公司酵母表达操作手册进行,略有改进。将筛选到的阳性酵母菌接种于20mLYPD培养基,30℃,300r/min培养至A600达到3~5,离心收集菌体转接于50mL BMMY培养基中,进行诱导表达。30℃,300r/min培养72h,期间每24h补加终浓度为0.5%(V/V)的甲醇。诱导后,12000r/min离心15min收集培养液上清,进行Tricine-SDS-PAGE,结果见图1,可以看到,随着发酵时间的延长,2.1kD初条带明显增加,表明发酵产生杂合肽。
实施例2重组杂合肽Melectin-P最小抑菌浓度(MIC)测定
本实施例对杂合肽Melectin-P同原始抗菌肽Melectin、Protonectin的抑菌活性进行了比较。其中原始抗菌肽Melectin的氨基酸序列为GFLSILKKVLPKVMAHMK(SEQ ID NO:3);原始抗菌肽Protonectin的氨基酸序列为ILGTILGLLKGL(SEQ ID NO:4)。
采用微量肉汤稀释法测定重组Melectin-P抑菌活性。0.02%乙酸溶液(含0.4%牛血清白蛋白)稀释重组杂合肽,得到2倍梯度稀释抗菌肽贮存液(2560,1280,…,20和10μg/mL),存放于聚丙烯离心管中。测试菌接种于MHA培养基中过夜培养,将测试菌过夜培养物稀释至2×105~7×105cfu/mL,分别取100μL加到96孔细胞培养板每行1~10号孔,将按梯度稀释抗菌肽10μL对应加到1~9号孔,10号孔不加抗菌肽,作为阳性对照,11号孔加入100μL新鲜MHB培养基作为空白对照,37℃培养18至24h后,用酶标仪测490nm吸光值。吸光度比10号孔低50%以上孔内浓度即定义为对该测试菌最小抑菌浓度MIC,其中对杂合肽Melectin-P同原始抗菌肽Melectin、Protonectin的抑菌活性进行了比较,结果见表2。
表2杂合抗菌肽的最小抑菌浓度(MIC)
由表2可知,杂合肽Melectin-P对大肠杆菌、肠道沙门氏菌和鼠伤寒沙门氏菌的MIC分别为4.6μg/ml、2.9μg/ml和9.3μg/ml,显著低于原始抗菌肽Protonectin和Melectin;杂合肽Melectin-P对铜绿假单胞菌的MIC为5.4μg/ml,显著低于原始抗菌肽Protonectin和Melectin。杂合肽Melectin-P对金黄色葡萄球菌的MIC为9.1μg/ml,同样显著低于原始抗菌肽Protonectin和Melectin。因此,杂合抗菌肽在抑菌能力方面较原始抗菌肽更优,对常见致病菌具有较好的抑菌活性。
实施例3重组杂合肽Melectin-P热稳定性和酸碱稳定性检测
1)热稳定检测将杂合抗菌肽Melectin-P分别煮沸5、10、15、20、25、30、35、40、45min,再以金黄色葡萄球菌为指示剂进行抑菌试验,24h后测量抑菌圈大小,结果见图2,可以看到其抑菌性能非常稳定。
2)同时,配置pH 3.0~11.0的缓冲液,分别加入20μL杂合抗菌肽至不同pH缓冲液,以不加抗菌肽的不同pH缓冲液为对照,做抑菌试验,结果见图3,可以看出,无论是在酸性环境还是碱性环境,杂合抗菌肽的抑菌性能基本保持稳定,应用范围广泛。
实施例4重组杂合肽Melectin-P对仔猪生长性能及腹泻率的影响
本实施例分别于试验开始和结束时,晨饲前空腹称重,记录仔猪每天的耗料量及腹泻情况,以重复为单位计算耗料量,并计算各组仔猪平均日增重、平均日采食量、料重比及腹泻率。腹泻率(%)=试验全期腹泻头次/(试验头数×试验天数)×100%。
实验方法:试验采用单因子试验设计,选用120头28日龄平均体重为(7.06±1.12)kg的健康“杜×长×大”三元杂交断奶仔猪,按照相近体重、公母各半的原则随机分为4组,每组5个重复,每个重复6头猪。具体分组见表3。其中基础饲粮参照NRC(2012)饲养标准配制,其组成及营养水平见表4。
表3日粮结构
组别 | 日粮结构 | 抗菌肽添加量 |
实验组 | 基础日粮+杂合肽Melectin-P | 0.2% |
对照组一 | 基础日粮 | 0% |
对照组二 | 基础日粮+抗菌肽Protonectin | 0.2% |
对照组三 | 基础日粮+抗菌肽Melectin | 0.2% |
表4基础日粮组成及营养水平(风干基础)
注:预混料为每千克日粮提供:维生素A 10000IU;维生素D3 3500IU;维生素E40mg;维生素K3 3.0mg;维生素B1 1.5mg;维生素B2 8.0mg;维生素B6 4.0mg;维生素B120.025mg;叶酸1.2mg;生物素0.15mg;烟酸50mg;泛酸20mg;铜50mg;铁135mg;锌140mg;锰50mg;硒0.36mg。营养水平为计算值。
试验期间严格按照猪场规定,统一进行驱虫。每天饲喂3次,保证每次采食后料槽有少许余料,所有猪仔自由采食和饮水。保持猪舍卫生清洁和空气流通,定期对猪圈进行清扫和消毒。每天观察仔猪情况,记录仔猪的采食、精神状况及有无腹泻。试验期30d。
生长性能及腹泻率:分别于试验开始和结束时,晨饲前空腹称重,记录仔猪每天的耗料量及腹泻情况,以重复为单位计算耗料量,并计算各组仔猪平均日增重、平均日采食量、料重比及腹泻率。腹泻率(%)=试验全期腹泻头次/(试验头数×试验天数)×100%。
结果见表5。
表5不同处理组对断奶仔猪生长性能与腹泻率的影响
指标 | 实验组 | 对照组一 | 对照组二 | 对照组三 |
平均日增重/(g/d) | 293.36 | 253.32 | 284.28 | 289.14 |
平均日采食量/(g/d) | 486.23 | 441.23 | 480.81 | 485.27 |
料重比 | 1.66 | 1.74 | 1.69 | 1.68 |
腹泻率/% | 2.53 | 9.27 | 7.25 | 6.29 |
由表5可知,与对照组相比,实验组平均日增重提高15.8%,同原始抗菌肽组相比也分别提升了3.2%和1.46%。
与对照组一相比,实验组平均日采食量提高10.2%,同原始抗菌肽组(对照组二、对照组三)相比也分别提升了1.1%和0.2%。
与对照组一相比,实验组平均料重比降低4.6%,同原始抗菌肽组(对照组二、对照组三)相比也分别降低了1.8%和1.2%。
与对照组一相比,实验组腹泻率降低6.74个百分点,同原始抗菌肽组(对照组二、对照组三)相比也分别降低了4.72%和3.76%。
综合来看,杂合肽Melectin-P对仔猪平均日增重、平均日采食量、料重比及腹泻率等仔猪生长指标改善显著,且优于原始抗菌肽组。
实施例5重组杂合肽Melectin-P对血清免疫指标的影响
采用ELISA试剂盒法测定血清中IgA、IgG、IgM、IL-2、IL-4、IL-8和TNF-α含量。
于实施例4试验结束后,每个重复随机选取2头仔猪,前腔静脉采血10mL置于未加抗凝剂的试管中,放置30min后,4000r/min离心10min,分离血清,分装成2份,-20℃保存。采用ELISA试剂盒法测定血清中IgA、IgG、IgM含量。结果见表6。
表6抗菌肽对断奶仔猪血清免疫指标的影响
指标 | 实验组 | 对照组一 | 对照组二 | 对照组三 |
IgA/(mg/mL) | 564.1 | 525.4 | 557.2 | 546.3 |
IgG/(mg/mL) | 8.20 | 7.25 | 7.64 | 7.58 |
IgM/(mg/mL) | 213.7 | 167.32 | 188.8 | 186.2 |
由表6可知,与对照组一相比,实验组仔猪血清IgA含量提升7.36%,同原始抗菌肽组(对照组二、对照组三)相比也分别提升了1.24%和3.25%。
与对照组一相比,实验组血清IgG含量提升13.1%,同原始抗菌肽组(对照组二、对照组三)相比也分别提升了7.33%和8.18%。
与对照组一相比,实验组血清IgM含量提升27.72%,同原始抗菌肽组(对照组二、对照组三)相比也分别降低了13.19%和14.77%。
综合来看,杂合肽Melectin-P对仔猪血清IgA、IgG、IgM含量指标改善显著,且优于原始抗菌肽组。
综上所述,本实施例1成功构建了基因工程菌P.pastoris X-33/pPICZα-A-Melectin-P,杂合肽具有广谱抗菌活性,对革兰氏阳性菌及革兰氏阴性菌均有不同程度的抑菌活性。在仔猪日粮中添加杂合抗菌肽Melectin-P可提升仔猪平均日增重、提升平均日采食量、降低料重比并降低断奶仔猪腹泻率,同时提高机体免疫指标。使其在食品防腐、疾病防治和动物饲料添加剂等方面显示很好的应用前景。
上述具体实施方式对本发明作了详细说明,但是本发明不限于上述实施例,在所属技术领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下作出各种变化。此外,在不冲突的情况下,本发明的实施例及实施例中的特征可以相互组合。
SEQUENCE LISTING
<110> 江苏尚宝牧业有限公司、广州格拉姆生物科技有限公司
<120> 一种杂合抗菌肽及其制备方法和应用
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> PRT
<213> 人工序列
<400> 1
Gly Thr Ile Leu Gly Leu Leu Lys Lys Val Leu Pro Lys Val Met Ala
1 5 10 15
His Met Lys
<210> 2
<211> 57
<212> DNA
<213> 人工序列
<400> 2
ggtactattt tgggactgtt gaagaaagtg ctgccaaagg tcatggctca catgaaa 57
<210> 3
<211> 18
<212> PRT
<213> 人工序列
<400> 3
Gly Phe Leu Ser Ile Leu Lys Lys Val Leu Pro Lys Val Met Ala His
1 5 10 15
Met Lys
<210> 4
<211> 12
<212> PRT
<213> 人工序列
<400> 4
Ile Leu Gly Thr Ile Leu Gly Leu Leu Lys Gly Leu
1 5 10
Claims (12)
1.杂合抗菌肽,其氨基酸序列如SEQ ID NO:1所示。
2.一种核酸分子,编码权利要求1所述杂合抗菌肽。
3.根据权利要求2所述的核酸分子,其特征在于,所述核酸分子的序列如SEQ ID NO:2所示。
4.一种表达载体,包含权利要求2~3任一项所述核酸分子。
5.一种重组细胞,包含权利要求4所述表达载体。
6.权利要求1所述杂合抗菌肽或权利要求2~3任一项所述核酸分子或权利要求4所述表达载体或权利要求5所述重组细胞在制备微生物抑制剂中的应用。
7.根据权利要求6所述的应用,其特征在于,所述微生物为大肠杆菌、沙门氏菌、铜绿假单胞菌或金黄色葡萄球菌。
8.权利要求1所述杂合抗菌肽或权利要求2~3任一项所述核酸分子或权利要求4所述表达载体或权利要求5所述重组细胞在制备添加剂中的应用。
9.根据权利要求8所述的应用,其特征在于,所述添加剂为防腐剂或药品。
10.根据权利要求9所述的应用,其特征在于,所述药为兽药。
11.根据权利要求8所述的应用,其特征在于,所述添加剂为食品添加剂、饲料添加剂、化妆品添加剂或卫生产品添加剂。
12.一种添加剂,包含权利要求1所述杂合抗菌肽。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110893482.8A CN113801198B (zh) | 2021-08-04 | 2021-08-04 | 一种杂合抗菌肽及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110893482.8A CN113801198B (zh) | 2021-08-04 | 2021-08-04 | 一种杂合抗菌肽及其制备方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113801198A CN113801198A (zh) | 2021-12-17 |
CN113801198B true CN113801198B (zh) | 2023-06-27 |
Family
ID=78893255
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110893482.8A Active CN113801198B (zh) | 2021-08-04 | 2021-08-04 | 一种杂合抗菌肽及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113801198B (zh) |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009109156A2 (en) * | 2008-03-04 | 2009-09-11 | Ustav Organicke Chemie A Bioch | Novel antimicrobial peptides and their application |
CN103205370A (zh) * | 2012-05-15 | 2013-07-17 | 广州格拉姆生物科技有限公司 | 一种杂合抗菌肽毕赤酵母工程菌制备方法及应用 |
CN103429621A (zh) * | 2011-01-07 | 2013-12-04 | 威联生物制药私人有限公司 | 抗微生物融合化合物及其用途 |
CA2877272A1 (en) * | 2012-06-26 | 2014-01-03 | Biovalence Sdn. Bhd. | Dosage regime of fusion compounds |
CN108690140A (zh) * | 2018-04-17 | 2018-10-23 | 广州格拉姆生物科技有限公司 | 一种杂合肽及其在抑菌中的应用 |
CN108752431A (zh) * | 2018-04-16 | 2018-11-06 | 广州格拉姆生物科技有限公司 | 杂合抗菌肽Mel-MytB及其应用 |
-
2021
- 2021-08-04 CN CN202110893482.8A patent/CN113801198B/zh active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009109156A2 (en) * | 2008-03-04 | 2009-09-11 | Ustav Organicke Chemie A Bioch | Novel antimicrobial peptides and their application |
CN103429621A (zh) * | 2011-01-07 | 2013-12-04 | 威联生物制药私人有限公司 | 抗微生物融合化合物及其用途 |
CN103205370A (zh) * | 2012-05-15 | 2013-07-17 | 广州格拉姆生物科技有限公司 | 一种杂合抗菌肽毕赤酵母工程菌制备方法及应用 |
CA2877272A1 (en) * | 2012-06-26 | 2014-01-03 | Biovalence Sdn. Bhd. | Dosage regime of fusion compounds |
CN108752431A (zh) * | 2018-04-16 | 2018-11-06 | 广州格拉姆生物科技有限公司 | 杂合抗菌肽Mel-MytB及其应用 |
CN108690140A (zh) * | 2018-04-17 | 2018-10-23 | 广州格拉姆生物科技有限公司 | 一种杂合肽及其在抑菌中的应用 |
Non-Patent Citations (3)
Title |
---|
Melectin: A Novel Antimicrobial Peptide from the Venom of the Cleptoparasitic Bee Melecta albifrons;V .Cˇerˇovsky et al.;《ChemBioChem 》;第9卷;第2815 – 2821页 * |
Membrane Perturbation Action Mode and Structure-Activity Relationships of Protonectin, a Novel Antimicrobial Peptide from the Venom of the Neotropical Social Wasp Agelaia pallipes pallipes;Kairong Wang et al.;《Antimicrobial Agents and Chemotherapy 》;第57卷(第10期);第4632–4639页 * |
杂合抗菌肽设计及生物学活性的研究进展;武如娟等;《中国生物工程杂志》;第33卷(第9期);第94-110页 * |
Also Published As
Publication number | Publication date |
---|---|
CN113801198A (zh) | 2021-12-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN103429093A (zh) | 饲料添加剂组合物 | |
CN107164344B (zh) | 一类耐热型植酸酶突变体及其编码基因和应用 | |
CN110218245B (zh) | 一种具有抑菌活性的抗菌肽atmp7及其在用于制备抗菌剂中的应用 | |
CN112011481A (zh) | 一株防治畜禽细菌性腹泻的罗伊氏乳杆菌及其应用 | |
CN102559539A (zh) | 嗜酸乳杆菌及其应用与饲料添加剂和预混料 | |
CN102250817A (zh) | 一株饲用凝结芽孢杆菌及其应用 | |
Dai et al. | Effects of antimicrobial peptide Microcin C7 on growth performance, immune and intestinal barrier functions, and cecal microbiota of broilers | |
CN102559816A (zh) | 一种抗菌脂肽的制备方法及其在兽医药中的应用 | |
CN113025534B (zh) | 一株具有较强抑菌作用的新种芽孢杆菌x901株及其应用 | |
CN113801198B (zh) | 一种杂合抗菌肽及其制备方法和应用 | |
JP6523966B2 (ja) | 大腸菌性下痢症の予防 | |
KR101247687B1 (ko) | 항생제 대체용 p5 펩타이드를 함유하는 사료첨가제 및 그의 제조방법 | |
WANG et al. | Transgenic rice expressing a novel phytase-lactoferricin fusion gene to improve phosphorus availability and antibacterial activity | |
CN114908009B (zh) | 一株口粘液乳杆菌pr63及其应用 | |
CN117003828A (zh) | 抗菌肽突变体及其应用 | |
CN105646718A (zh) | 一种m1型巨噬细胞激活肽与il-2融合蛋白、融合基因、表达载体及其构建方法 | |
CN114891678A (zh) | 多粘芽孢杆菌cpl258及其筛选和应用 | |
JP2004536589A (ja) | ウシ上皮成長因子の核酸及びタンパク質配列 | |
KR101300218B1 (ko) | 항생제 대체용 a3 펩타이드를 함유하는 사료첨가제 및 그의 제조 방법 | |
Bishehkolaei et al. | Milk kefir as a natural probiotic, individually or in combination with organic acids in broiler chickens: influence on the immune-related gene expression, intestinal morphology, microbiota activity, and serum biochemistry. | |
CN114933644B (zh) | 一种泥鳅抗菌肽Ma-sHep及其应用 | |
CN117106640B (zh) | 一种高效降解霉菌毒素的生物制剂及其制备方法 | |
CN115851556B (zh) | 一种非解乳糖链球菌及其应用 | |
CN114369143B (zh) | 一种生物活性多肽及其应用 | |
CN116056587A (zh) | 一种作为改善生长表现和免疫反应的饲料补充物的重组抗微生物胜肽 |
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 |