CN115850427B - Conus double-alpha helix antibacterial peptide, preparation method and application thereof - Google Patents
Conus double-alpha helix antibacterial peptide, preparation method and application thereof Download PDFInfo
- Publication number
- CN115850427B CN115850427B CN202211585193.2A CN202211585193A CN115850427B CN 115850427 B CN115850427 B CN 115850427B CN 202211585193 A CN202211585193 A CN 202211585193A CN 115850427 B CN115850427 B CN 115850427B
- Authority
- CN
- China
- Prior art keywords
- antibacterial peptide
- mol
- antibacterial
- cono
- double
- 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 147
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 241000237970 Conus <genus> Species 0.000 title description 7
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 52
- 238000000034 method Methods 0.000 claims abstract description 17
- 239000003814 drug Substances 0.000 claims abstract description 11
- 241000894006 Bacteria Species 0.000 claims abstract description 10
- 238000012216 screening Methods 0.000 claims abstract description 7
- 241000233866 Fungi Species 0.000 claims abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 17
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 68
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 57
- 150000001413 amino acids Chemical class 0.000 claims description 50
- 108090000623 proteins and genes Proteins 0.000 claims description 35
- 229920001184 polypeptide Polymers 0.000 claims description 34
- 235000018102 proteins Nutrition 0.000 claims description 21
- 102000004169 proteins and genes Human genes 0.000 claims description 21
- 108700042778 Antimicrobial Peptides Proteins 0.000 claims description 18
- 102000044503 Antimicrobial Peptides Human genes 0.000 claims description 18
- 241000235058 Komagataella pastoris Species 0.000 claims description 18
- 101800001442 Peptide pr Proteins 0.000 claims description 18
- 241000588724 Escherichia coli Species 0.000 claims description 12
- 230000000843 anti-fungal effect Effects 0.000 claims description 11
- 229940121375 antifungal agent Drugs 0.000 claims description 11
- 239000002435 venom Substances 0.000 claims description 10
- 231100000611 venom Toxicity 0.000 claims description 10
- 210000001048 venom Anatomy 0.000 claims description 10
- 241000194042 Streptococcus dysgalactiae Species 0.000 claims description 9
- 239000003242 anti bacterial agent Substances 0.000 claims description 9
- 244000005700 microbiome Species 0.000 claims description 9
- 229940115920 streptococcus dysgalactiae Drugs 0.000 claims description 9
- 239000000126 substance Substances 0.000 claims description 9
- 238000003786 synthesis reaction Methods 0.000 claims description 9
- 239000000654 additive Substances 0.000 claims description 7
- 230000000996 additive effect Effects 0.000 claims description 7
- 235000001014 amino acid Nutrition 0.000 claims description 5
- 108010026552 Proteome Proteins 0.000 claims description 4
- 238000004364 calculation method Methods 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 235000018417 cysteine Nutrition 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 108020004705 Codon Proteins 0.000 claims description 3
- 150000001768 cations Chemical class 0.000 claims description 3
- 150000001945 cysteines Chemical class 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 14
- 229940079593 drug Drugs 0.000 abstract description 4
- 238000011161 development Methods 0.000 abstract description 3
- 230000005764 inhibitory process Effects 0.000 abstract description 3
- 241000192125 Firmicutes Species 0.000 abstract 1
- 229940124350 antibacterial drug Drugs 0.000 abstract 1
- 239000002243 precursor Substances 0.000 description 19
- 230000012010 growth Effects 0.000 description 11
- 239000000047 product Substances 0.000 description 9
- 230000002401 inhibitory effect Effects 0.000 description 8
- 239000002773 nucleotide Substances 0.000 description 8
- 125000003729 nucleotide group Chemical group 0.000 description 8
- 241000235648 Pichia Species 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 101710117064 Trimethylamine corrinoid protein 1 Proteins 0.000 description 5
- 229940088710 antibiotic agent Drugs 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 229940024606 amino acid Drugs 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 231100000614 poison Toxicity 0.000 description 4
- 241000894007 species Species 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 101100379209 Arabidopsis thaliana APD3 gene Proteins 0.000 description 3
- 241000237858 Gastropoda Species 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- 241000257303 Hymenoptera Species 0.000 description 3
- 108050003126 conotoxin Proteins 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000002574 poison Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- 241000252085 Anguilla rostrata Species 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 241000270295 Serpentes Species 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 125000002091 cationic group Chemical group 0.000 description 2
- 238000011097 chromatography purification Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000012969 defense response to bacterium Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229940126585 therapeutic drug Drugs 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 241000239290 Araneae Species 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000186146 Brevibacterium Species 0.000 description 1
- 241000258920 Chilopoda Species 0.000 description 1
- 241000032572 Conus betulinus Species 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241000191938 Micrococcus luteus Species 0.000 description 1
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 241000239226 Scorpiones Species 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 241000193985 Streptococcus agalactiae Species 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 101710099833 Venom protein Proteins 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000004422 calculation algorithm Methods 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- -1 instruments Substances 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010831 paired-sample T-test Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a conoid double alpha-helix antibacterial peptide, the amino acid sequence of which is one of SEQ ID NO. 1-4, and a method for screening and obtaining the conoid-derived antibacterial peptide and application of the obtained antibacterial peptide in preparation of antibacterial products or medicines. The conoid double alpha-helix antibacterial peptide provided by the invention has broad-spectrum antibacterial activity, has strong inhibition effects on gram-positive bacteria, gram-negative bacteria and common fungi, and has important effects and values in the development and application of antibacterial drugs or biological products.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a conoid double-alpha helical antibacterial peptide, a preparation method and application thereof.
Background
Conus is a carnivorous marine gastropoda mollusk, known for its beautiful coat and special toxins secreted in the body. Conoids are registered in about 1000 species in the world's marine species and they generally live in tropical and subtropical intertidal zones in many parts of the world. Hundreds of conotoxin peptides can be produced in the venom of the conotoxin and can be used for predating various animals including worms, snails or fish. Over the last few decades, over 70000 natural conotoxins have been found, widely used in pharmacological and neuroscience research. The novel drug is a new attractive drug resource because of novel chemical structure, strong biological activity and high targeting selectivity.
The problem of bacterial resistance is becoming more and more serious due to the abuse of traditional antibiotics, and it is becoming more and more difficult to find new antibiotics. Antibacterial peptides are host defensive peptides, most of which are cationic (positively charged) and amphiphilic (hydrophilic and hydrophobic) alpha helix/beta sheet peptide molecules. Based on membrane permeability, the cationic antibacterial peptide can be combined with and interacted with negatively charged cell membranes to change electrochemical potential on the cell membranes, induce cell membrane damage to permeate larger molecules such as proteins, destroy cell morphology and cell membranes, and finally lead to cell death. Compared with the traditional antibiotics, the antibacterial peptides have wide antibacterial activity, including antibacterial, antifungal, antiviral and anticancer activities, even overcome the drug resistance of bacteria, and are the biological resource with the most potential to replace the antibiotics at present.
Antibacterial peptides are the first line of defense against exogenous pathogens as an important component of the innate immune defense system. Because of its broad-spectrum antimicrobial activity, rapid synthesis after infection, and difficulty in developing drug resistance, has been widely studied. The complex multi-component venom contains various bioactive peptides, including antibacterial peptides, and has been found in various poisonous animals such as spiders, snakes, scorpions, bees and the like, and has good development prospect as a therapeutic drug.
Although more than 3000 antimicrobial peptides have been identified to date from animals, fungi, plants and bacteria. A large number of biologically active antimicrobial peptides are also found in a variety of venom-producing animals. 15 venom short peptide sequences from 5 ants were reported to have antibacterial activity. 13 venom short peptide sequences from 7 bees were reported to have antibody activity. 10 venom short peptide sequences from 9 snakes were reported to have antibody activity. 55 active antibacterial peptides were found from more than 30 centipedes. The reported antimicrobial peptides have mostly antibacterial activity, and partly antifungal or antimicrobial activity against other pathogenic microorganisms.
However, compared with animals such as worms, snails and fishes, the studies on antibacterial peptides in the conoids are very few, only a few antibacterial peptide sequences are reported at present, and the studies on antibacterial application of the conoids are very rare. One study reported previously that a connoxin from the cono 1 superfamily could inhibit the growth of mycobacterium tuberculosis. Another open-chain MVIIA structure derived from conoxin inhibits fungal growth. In the prior art, the problems of small number and single antibacterial species of the conoid-derived antibacterial peptide generally exist, and most of the conventional antibacterial peptides are obtained through blind screening, for example, the conventional antibacterial peptides are directly separated and extracted from organisms through experimental means, or the obtained antibacterial peptide gene sequences are cloned and then introduced into microorganisms for culturing, and then the obtained antibacterial peptides are further separated, purified and identified through a series of experimental means. For example, the preparation method of the marine organism antibacterial peptide disclosed in the Chinese patent document CN115232850A comprises the steps of freeze-drying and crushing marine organism materials, then carrying out enzymolysis, and separating and extracting the antibacterial peptide; in vitro recombinant expression and identification of novel American eel antibacterial peptide published by Chinese patent document CN115057918A, cloning an antibacterial domain gene of bactericidal protein from the American eel, constructing an expression vector, introducing into escherichia coli for expression, and further identifying after chromatographic purification by using a chromatographic purification column. The method has the advantages of more experimental procedures, complicated operation, long time consumption, low efficiency and high cost, and is not beneficial to development, research and application of the antibacterial peptide.
Therefore, how to quickly and effectively deeply dig the antibacterial function of the cono polypeptide, obtain more cono antibacterial peptides which are easy to obtain and have wide antibacterial species, provide more choices for replacing the traditional antibiotic therapeutic drugs, and have important research significance and potential market value.
Disclosure of Invention
In order to solve the technical problems, the invention provides a conomical double alpha helix antibacterial peptide, wherein the amino acid sequence of the antibacterial peptide is one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4.
Further, the nucleotide sequence of the amino acid sequence shown in SEQ ID NO. 1 is shown in SEQ ID NO. 5, the nucleotide sequence of the amino acid sequence shown in SEQ ID NO. 2 is shown in SEQ ID NO. 6, the nucleotide sequence of the amino acid sequence shown in SEQ ID NO. 3 is shown in SEQ ID NO. 7, and the nucleotide sequence of the amino acid sequence shown in SEQ ID NO. 4 is shown in SEQ ID NO. 8.
Further, the antimicrobial peptide comprises a double alpha helix structure.
Further, the antimicrobial peptide is capable of specifically inhibiting the growth of Pichia, pichia and Brevibacterium, pichia and Escherichia coli.
In another aspect of the invention, the invention also provides a precursor protein of the conomical double-alpha helical antibacterial peptide, wherein the amino acid sequence of the precursor protein of the conomical double-alpha helical antibacterial peptide is one of SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12, the precursor protein of the conomical double-alpha helical antibacterial peptide with the sequence of SEQ ID NO. 9 is determined according to the nucleotide sequence code shown in SEQ ID NO. 5, the precursor protein of the conomical double-alpha helical antibacterial peptide with the sequence of SEQ ID NO. 10 is determined according to the nucleotide sequence code shown in SEQ ID NO. 6, the precursor protein of the conouseful double-alpha helical antibacterial peptide with the sequence of SEQ ID NO. 11 is determined according to the nucleotide sequence code shown in SEQ ID NO. 7, and the precursor protein of the conouseful double-alpha helical antibacterial peptide with the sequence of SEQ ID NO. 12 is determined according to the nucleotide sequence code shown in SEQ ID NO. 8.
In another aspect of the present invention, a process for preparing a conomical double alpha-helical antibacterial peptide according to any one of claims 1 to 4, comprising the steps of:
1) Obtaining a multiple set of conoid data, the multiple set of conoid data comprising a conoid genome, a transcriptome and a venom proteome data set;
2) Identifying an antibacterial peptide precursor gene sequence from a conomic data set by adopting a homology comparison method;
3) Translating the identified antibacterial peptide precursor gene sequence into a conoid antibacterial peptide precursor protein based on a codon encoding rule to obtain an amino acid sequence of the conoid antibacterial peptide precursor protein;
4) Referring to the antibacterial peptide polypeptide fragment with known antibacterial activity, manually screening and designing the potential mature active peptide region of the obtained cono antibacterial peptide precursor protein amino acid sequence through physical and chemical property calculation and protein three-dimensional structure prediction to obtain the amino acid sequence of the cono antibacterial peptide;
5) Synthesizing polypeptide according to the designed amino acid sequence of the conoid antibacterial peptide by a chemical synthesis method;
6) And mixing and culturing the synthesized polypeptide with different microorganisms, and identifying the antibacterial effect of the polypeptide, thereby obtaining the conoid antibacterial peptide.
Specifically, in step 4), the potential mature active peptide region of the amino acid sequence of the obtained conoid antimicrobial peptide precursor protein is manually screened and designed, and the designed polypeptide meets the following conditions:
a. which is a cation with a positive charge,
b. the isoelectric point of the material is more than 8.0,
c. with CTDD and PAAC values greater than 0.5,
d. it has a double alpha-helical structure and,
e. the number of cysteines in the amino acid sequence is less than 5,
f. the amino acid sequence is not longer than 30 amino acids.
Specifically, in step 6), the synthesized polypeptide is mixed and cultured with different microorganisms including experimental E.coli, pichia and Streptococcus dysgalactiae.
In still another aspect, the invention further provides an application of the conoid-derived antibacterial peptide in preparation of antifungal and antibacterial products.
Further, the fungus is pichia pastoris, and the bacteria are streptococcus dysgalactiae and escherichia coli.
Further, the application includes at least one of the following applications:
(I) Preparing a bacteriostat;
(II) preparing a medicament;
(III) preparation of the additive.
Further, the antibacterial agent, the medicine and the additive all contain cono double alpha helix antibacterial peptide with the concentration of 97.5-780 mu mol/L and the amino acid sequence shown as SEQ ID NO. 1, cono double alpha helix antibacterial peptide with the concentration of 9.0-572 mu mol/L and the amino acid sequence shown as SEQ ID NO. 2, cono double alpha helix antibacterial peptide with the concentration of 46-367 mu mol/L and the amino acid sequence shown as SEQ ID NO. 3 or cono double alpha helix antibacterial peptide with the concentration of 183-366 mu mol/L and the amino acid sequence shown as SEQ ID NO. 4.
Preferably, in the antibacterial agent, the medicine and the additive, when the cono double alpha helix antibacterial peptide with the amino acid sequence shown as SEQ ID NO. 1 is selected to prepare the product for resisting the escherichia coli, the concentration of the antibacterial peptide is preferably 97.5 mu mol/L, 195 mu mol/L, 390 mu mol/L or 780 mu mol/L;
when the cono double alpha helix antibacterial peptide with the amino acid sequence shown as SEQ ID NO. 1 is selected to prepare the Pichia pastoris resistant product, the concentration of the antibacterial peptide is preferably 24.4 mu mol/L, 48.8 mu mol/L, 195 mu mol/L, 390 mu mol/L or 780 mu mol/L;
when the cono double alpha helix antibacterial peptide with the amino acid sequence shown as SEQ ID NO. 2 is selected to prepare the product for resisting streptococcus dysgalactiae, the concentration of the antibacterial peptide is preferably 17.9 mu mol/L, 35.8 mu mol/L, 71.5 mu mol/L, 143 mu mol/L, 286 mu mol/L or 572 mu mol/L;
when the cono double alpha helix antibacterial peptide with the amino acid sequence shown as SEQ ID NO. 2 is selected to prepare the Pichia pastoris resistant product, the concentration of the antibacterial peptide is preferably 9.0 mu mol/L, 17.9 mu mol/L, 35.8 mu mol/L, 71.5 mu mol/L, 143 mu mol/L or 286 mu mol/L;
when the cono double alpha helix antibacterial peptide with the amino acid sequence shown as SEQ ID NO. 3 is selected to prepare the Pichia pastoris resistant product, the concentration of the antibacterial peptide is preferably 11.5 mu mol/L, 23 mu mol/L, 46 mu mol/L, 92 mu mol/L, 184 mu mol/L or 367 mu mol/L;
when the cono double alpha helix antibacterial peptide with the amino acid sequence shown as SEQ ID NO. 4 is selected to prepare the Pichia pastoris resistant product, the concentration of the antibacterial peptide is preferably 183 or 366 mu mol/L.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, four conomical antibacterial peptides are excavated from a plurality of groups of conomical data, the effect of resisting three different fungi and bacteria is discovered for the first time, the antibacterial potential of the conomical polypeptide is emphasized, wherein, acipensen_6-1 and Acipensen_6-2 can inhibit the growth of fungus pichia pastoris, TCP-1 shows inhibition activity on escherichia coli and pichia pastoris, cbTbeta shows inhibition activity on streptococcus dysgalactiae and pichia pastoris, the variety and antibacterial variety of the conomical antibacterial peptides are enriched, and the conomical antibacterial peptide is hopeful to be applied to prepare products with antibacterial activity and replace antibiotics for treating fungus or bacterial infection in clinic, and has good application prospect and economic value.
2. The method is based on a plurality of groups of methods, a plurality of potential antibacterial peptide sequences are screened from a biological big data layer, and the antibacterial peptide with biological activity can be rapidly and efficiently identified by combining activity simulation prediction and in-vitro experiment verification.
3. The antibacterial peptide provided by the invention has a double alpha helical structure, and is good in amphiphilicity and good in binding capacity with a microbial membrane.
Drawings
FIG. 1 is a schematic diagram of the three-dimensional structure of an antibacterial peptide with the amino acid sequence of SEQ ID NO. 1-4;
FIG. 2 is a high performance liquid chromatogram of the antibacterial peptide with the amino acid sequence of SEQ ID NO. 1 after synthesis;
FIG. 3 is a mass spectrum diagram of the synthetic antibacterial peptide with the amino acid sequence of SEQ ID NO. 1;
FIG. 4 is a high performance liquid chromatogram of the antibacterial peptide with the amino acid sequence of SEQ ID NO. 2 after synthesis;
FIG. 5 is a mass spectrum diagram of the synthetic antibacterial peptide with the amino acid sequence of SEQ ID NO. 2;
FIG. 6 is a high performance liquid chromatogram of the antibacterial peptide with the amino acid sequence of SEQ ID NO. 3 after synthesis;
FIG. 7 is a mass spectrum diagram of the synthetic antibacterial peptide with the amino acid sequence of SEQ ID NO. 3;
FIG. 8 is a high performance liquid chromatogram of the antibacterial peptide with the amino acid sequence of SEQ ID NO. 4 after synthesis;
FIG. 9 is a mass spectrum diagram of the synthetic antibacterial peptide with the amino acid sequence of SEQ ID NO. 4;
FIG. 10 is a graph showing the effect of the antibacterial peptide with the amino acid sequence of SEQ ID NO. 1 on inhibiting the growth of escherichia coli and pichia pastoris;
FIG. 11 is a graph showing the effect of the antibacterial peptide with the amino acid sequence of SEQ ID NO. 2 on inhibiting the growth of streptococcus dysgalactiae and pichia;
FIG. 12 is a graph showing the effect of the antibacterial peptide with the amino acid sequence of SEQ ID NO. 3 on inhibiting the growth of Pichia pastoris;
FIG. 13 is a graph showing the effect of the antibacterial peptide with the amino acid sequence of SEQ ID NO. 4 on inhibiting the growth of Pichia pastoris.
Technical terminology
The shorthand notation used in the full-length amino acid sequences of the present invention is a common abbreviation for those skilled in the art, specifically, R is arginine (Arg), L is leucine (Leu), G is glycine (Gly), N is asparagine (Asn), C is cysteine (Cys), T is threonine (Thr), V is valine (Val), M is methionine (Met), A is alanine (Ala), K is lysine (Lys), F is phenylalanine (Phe), S is serine (Ser), P is proline (Pro), E is glutamic acid (Glu), I is isoleucine (Ile), Q is glutamine (Gln), and W is tryptophan (Trp).
Detailed Description
The following description of the present invention will be made clearly and fully, and it is apparent that the embodiments described are some, but not all, of the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Materials, instruments, reagents and the like used in the following examples are commercially available unless otherwise specified. The technical means used in the examples, unless otherwise specified, are conventional means well known to those skilled in the art.
The Chinese cone-shaped cono (Conus betulinus) selected by the invention is obtained from three markets of Hainan province.
The invention adopts a homologous comparison method to identify the encoding sequence of the antimicrobial peptide precursor gene from multiple groups of chemical data of the barrel-shaped conoids. The protein sequence in the public antibacterial peptide database APD3 is used as a reference sequence to obtain the antibacterial peptide precursor gene nucleic acid sequence of four barrel-shaped conoids.
The invention utilizes the nucleic acid sequence of antibacterial peptide precursor genes of four barrel-shaped conoids, refers to antibacterial peptide polypeptide fragments with known antibacterial activity, and designs potential mature active peptide areas through physicochemical property calculation and protein three-dimensional structure prediction. Four polypeptide sequences were chemically synthesized, and then an antibacterial activity verification experiment of the polypeptides was performed. It was found that the four polypeptides provided by the present invention can be regarded as four novel antibacterial peptides.
In the following examples, the antibacterial peptide having the amino acid sequence of SEQ ID NO. 1 is TCP-1, the antibacterial peptide having the amino acid sequence of SEQ ID NO. 2 is cbTbeta, the antibacterial peptide having the amino acid sequence of SEQ ID NO. 3 is Acipensen_6-1, and the antibacterial peptide having the amino acid sequence of SEQ ID NO. 4 is Acipensen_6-2, which are collectively described herein and will not be described in detail.
Example 1 acquisition of the precursor Gene DNA sequence and the precursor protein sequence of an antimicrobial peptide
The preparation of the cono double alpha helix antibacterial peptide is carried out according to the following steps:
1) Obtaining a multiple set of conoid data, the multiple set of conoid data comprising a conoid genome, a transcriptome and a venom proteome data set;
2) Identifying an antibacterial peptide precursor gene sequence from a conomic data set by adopting a homology comparison method;
3) Translating the identified antibacterial peptide precursor gene sequence into a conoid antibacterial peptide precursor protein based on a codon encoding rule to obtain an amino acid sequence of the conoid antibacterial peptide precursor protein;
4) Referring to the antibacterial peptide polypeptide fragment with known antibacterial activity, manually screening and designing the potential mature active peptide region of the obtained cono antibacterial peptide precursor protein amino acid sequence through physical and chemical property calculation and protein three-dimensional structure prediction to obtain the amino acid sequence of the cono antibacterial peptide;
5) Synthesizing polypeptide according to the designed amino acid sequence of the conoid antibacterial peptide by a chemical synthesis method;
6) And mixing and culturing the synthesized polypeptide with different microorganisms, and identifying the antibacterial effect of the polypeptide, thereby obtaining the conoid antibacterial peptide.
The specific implementation mode is as follows:
1. multiple study data set preparation
The multiple sets of mathematical data in this example include genomic, transcriptomic, and venom proteomic data sets of the barrel conch.
Wherein the NCBI accession number of the genome dataset is: gca_016801955.1.
The transcriptome dataset included transcriptome data for 5 samples, respectively: big: a poison tube of an individual conoid with a body length of 10 cm; middle: a poison tube of a medium-sized individual conoid with a body length of 8.7 cm; small: a poison tube of a small individual conoid with a body length of 6 cm; bulb: the toxic capsule of the medium-sized individual conoids; normalized: the above-mentioned homogenized dataset of medium individual conotoxin tubes. The NCBI SRA database accession number for the transcriptome dataset is: SRS1009725 through SRS1009729.
The PRIDE database accession number for the venom protein dataset is: PXD014892.
The protein coding region of each transcript was predicted using TransDecoder software and the transcript level of the gene was calculated using FPKM values. All protein coding sequences from the genome and transcriptome are translated into amino acids, creating a comprehensive protein set of the barrel-shaped conoids.
2. Screening of antimicrobial peptide precursor Gene DNA sequences from multiple sets of chemical data
2927 verified antibacterial peptides in the public antibacterial peptide database APD3 are used as reference sequences.
Firstly, establishing an index library for sequence comparison by using makeblastdb in Blast software package by utilizing genes annotated by barrel-shaped conoid genome, wherein the command is makeblastdb-dbtype nucleic-parameter_seqids-in Conus.gene.cds. The potential AMP precursor genes were then predicted from the genome by using TBLASTN algorithm (e value less than 1 e-5) in Blast software, with sequences with alignment ratios greater than 50% retained, commanded as TBLASTN-query APDeu.fa-db Conus.gene.cds-outfmt 6-value 1e-5-out APDeu.fa.m8. And logging in windows visualization software AMPml software (2021, china,2021S R0424886, https:// gitub.com/flystar 233/AMPml) to upload potential AMP precursor gene files, selecting a CTDD and PAAC module, carrying out activity prediction on a section containing potential antibacterial peptide protein sequences in the reserved sequences, and screening out alignment sequences larger than 0.5 in the CTDD and PAAC models.
The same alignment method was used to predict antimicrobial peptide precursor genes for the transcript datasets of the five transcriptomes.
An index library is then created using the antimicrobial peptide precursor protein common to both the genomic and transcriptome datasets as a reference sequence. The proteome data are compared to the index library by using Blastp software, and the final consensus antibacterial peptide sequence set of three histology data sets is obtained, wherein the sequences with the comparison length of more than 50% and the comparison rate of more than 80% are reliable sequences.
Finally, the DNA sequences of four potential antimicrobial peptide precursor genes were obtained from genomic, transcriptome and proteomic datasets as shown in SEQ ID NOs 5-8. The antimicrobial peptide precursor gene is translated into a corresponding precursor protein sequence by using transcription rules, and is shown as SEQ ID NO. 9-12.
Example Conus tubulosis antibacterial peptide Activity Structure prediction
According to the four antimicrobial peptide precursor protein sequences identified from the multiple sets of chemical data in example one, the design of the potential mature active peptide region was performed with reference to the known antimicrobial peptide polypeptide sequences in the public antimicrobial peptide database APD3, as shown in SEQ ID NOS: 1-4.
Physical and chemical properties of the antimicrobial peptides, including molecular weight, isoelectric point, net charge value, and average hydrophilicity, were predicted using the ProtParam tool as shown in table 1 below:
table 1: physical and chemical property prediction method for four antibacterial peptides containing double alpha helical structures of barrel-shaped conoids
In order to ensure that the polypeptide has high possibility of having antibacterial activity, the designed polypeptide meets the following conditions: a. the polypeptide is a positive-charged cation; b. isoelectric point is greater than 8.0; ctdd and PAAC values greater than 0.5; d. has a double alpha helical structure; e. the number of cysteines in the sequence is less than 5; f. the sequence is no more than 30 amino acids in length.
Then, the three-dimensional structure of the four polypeptides described above was constructed using software PEP-FOLD3 suitable for the three-dimensional structure construction of short peptides, as shown in FIG. 1. Based on the construction of a three-dimensional model, four synthetic antibacterial peptides all contain double alpha helical structures.
Example three-barrel Conus antibacterial peptide Activity assay
1. Chemical synthesis:
the complete sequence is synthesized by adopting a standard amino acid polypeptide solid-phase synthesis method and is customized by Shanghai Jier Biochemical Co. The synthesized polypeptide product was purified using a high performance liquid chromatography system (HPLC) and then eluted with a 1mL/min acetonitrile gradient. As shown in FIGS. 2, 4, 6 and 8, the purity of the powder of the TCP-1, cbTbeta, acipensin _6-1 and Acipensen_6-2 polypeptides measured by HPLC-MS/MS was above 95%, and as shown in FIGS. 3, 5, 7 and 9, the molecular weights of the synthesized linear peptides were 2562.91, 3497.95, 2727.17 and 2731.2 daltons (Da), respectively. Finally, the mixture is stored in sterile deionized water at the temperature of minus 80 ℃.
2. Activity test:
1) The synthesized polypeptides (2 mg each) were dissolved in Phosphate Buffer (PBS) for multiple gradient dilutions, and prepared for use in multiple concentrations.
2) The culture experiment is carried out by using escherichia coli, micrococcus luteus, streptococcus dysgalactiae and pichia for standby.
3) After each strain was cultured to logarithmic growth phase, it was centrifuged and eluted 3 times with PBS.
4) All microorganisms were resuspended in PBS buffer (104 CFU/mL).
5) 10. Mu.L of the test bacterial suspension and 10. Mu.L of the PBS polypeptide solution sample were mixed in a 200. Mu.L tube, and the mixture was subjected to stationary culture at 37℃for 2 hours. PBS buffer served as a blank.
In the bacteriostasis assay, the mixture was transferred to 96-well enzyme-labeled wells, each well containing 200 μl of LB medium. All wells of the ELISA plate were placed in a 37℃microbiological incubator for cultivation. OD600 values were measured every half hour for a total of 20 hours (bacterial) or 48 hours (fungal) and used to plot the growth curve for the detected microorganisms. Three parallel wells were set up for each polypeptide and the experiment was repeated at least twice to obtain reliable results.
All data were counted using SPSS (Statistical Package for Social Sciences) and GraphPad Prism software and expressed as mean ± standard deviation (n=3). The significance of the differences between groups was tested using paired sample t-test and multiple comparisons.
Four polypeptides were identified to exhibit inhibitory effects on various microorganisms, and the antibacterial activity results are shown in table 2 below:
table 2: antibacterial activity of four antibacterial peptides containing double alpha helical structures of barrel-shaped cono
Note that: "N" represents no antibacterial activity
As shown in fig. 10-13, each of the four polypeptides inhibited growth of the fungus pichia pastoris. In addition, two antifungal antibacterial peptides (cbTbeta and TCP-1) also have inhibitory effects on the growth of different bacteria. CbTbeta can inhibit gram positive bacteria streptococcus agalactiae, and achieves the optimal antibacterial effect at 572 mu M. TCP-1 has remarkable antibacterial capacity on gram-negative bacteria such as escherichia coli, and the minimum antibacterial concentration is 97.5 mu M. Therefore, the conoid-derived antibacterial peptide provided by the invention has general antifungal capability and has the capability of specifically inhibiting gram-positive or gram-negative bacteria.
In summary, the above embodiments are only preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, but any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (16)
1. The cono double alpha helix antibacterial peptide is characterized in that the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4.
2. The conomical double alpha helical antibacterial peptide of claim 1, wherein the antibacterial peptide comprises a double alpha helical structure.
3. A method for preparing the cono double-alpha-helix antibacterial peptide, which is characterized by comprising the following specific steps of:
1) Obtaining a multiple set of conoid data, the multiple set of conoid data comprising a conoid genome, a transcriptome and a venom proteome data set;
2) Identifying an antibacterial peptide precursor gene sequence from a conomic data set by adopting a homology comparison method;
3) Translating the identified antibacterial peptide precursor gene sequence into a conoid antibacterial peptide precursor protein based on a codon encoding rule to obtain an amino acid sequence of the conoid antibacterial peptide precursor protein;
4) Referring to the antibacterial peptide polypeptide fragment with known antibacterial activity, manually screening and designing the potential mature active peptide region of the obtained cono antibacterial peptide precursor protein amino acid sequence through physical and chemical property calculation and protein three-dimensional structure prediction to obtain the amino acid sequence of the cono antibacterial peptide;
5) Synthesizing polypeptide according to the designed amino acid sequence of the conoid antibacterial peptide by a chemical synthesis method;
6) And mixing and culturing the synthesized polypeptide with different microorganisms, and identifying the antibacterial effect of the polypeptide, thereby obtaining the conoid antibacterial peptide.
4. The method for preparing a conomical double alpha-helical antibacterial peptide according to claim 3, wherein in the step 4), the potential mature active peptide region of the amino acid sequence of the obtained conomical antibacterial peptide precursor protein is artificially screened and designed, and the designed polypeptide meets the following conditions:
a. which is a cation with a positive charge,
b. the isoelectric point of the material is more than 8.0,
c. with CTDD and PAAC values greater than 0.5,
d. it has a double alpha-helical structure and,
e. the number of cysteines in the amino acid sequence is less than 5,
f. the amino acid sequence is not longer than 30 amino acids.
5. A process for the preparation of the cono double alpha helical antibacterial peptide of claim 3, wherein in step 6), the synthesized polypeptide is mixed and cultured with different microorganisms including experimental escherichia coli, pichia pastoris and streptococcus dysgalactiae.
6. Use of a conomical double alpha helical antimicrobial peptide according to any of claims 1 or 2 for the preparation of an antifungal product, wherein the fungus is pichia pastoris.
7. The application of the cono double-alpha helix antibacterial peptide in preparing an antibacterial product is characterized in that the amino acid sequence of the cono double-alpha helix antibacterial peptide is shown as SEQ ID NO. 2, and the bacterium is streptococcus dysgalactiae.
8. The application of the cono double-alpha-helix antibacterial peptide in preparing an antibacterial product is characterized in that the amino acid sequence of the cono double-alpha-helix antibacterial peptide is shown as SEQ ID NO. 1, and the bacterium is escherichia coli.
9. Use of a conomical double alpha helical antibacterial peptide according to claim 6 for the preparation of antifungal products, wherein said use comprises at least one of the following applications:
(I) Preparing a bacteriostat;
(II) preparing a medicament;
(III) preparation of the additive.
10. Use of a conomical double alpha helical antibacterial peptide according to any of claims 7 or 8 for the preparation of antibacterial products, wherein the use comprises at least one of the following applications:
(I) Preparing a bacteriostat;
(II) preparing a medicament;
(III) preparation of the additive.
11. The use of the cono double alpha-helix antibacterial peptide according to claim 9 for preparing antifungal products, wherein the concentration of the antibacterial peptide is 24.4 mu mol/L, 48.8 mu mol/L, 195 mu mol/L, 390 mu mol/L or 780 mu mol/L when the cono double alpha-helix antibacterial peptide with the amino acid sequence shown as SEQ ID NO. 1 is selected as the antibacterial peptide for preparing the Pichia pastoris resistant products.
12. The use of the cono double alpha-helix antibacterial peptide according to claim 9 for preparing antifungal products, wherein the concentration of the antibacterial peptide is 9.0 mu mol/L, 17.9 mu mol/L, 35.8 mu mol/L, 71.5 mu mol/L, 143 mu mol/L or 286 mu mol/L when the cono double alpha-helix antibacterial peptide with the amino acid sequence shown in SEQ ID NO. 2 is selected as the antibacterial peptide for preparing the Pichia pastoris.
13. The use of the cono double alpha-helix antibacterial peptide according to claim 9 for preparing antifungal products, wherein the concentration of the antibacterial peptide is 11.5 mu mol/L, 23 mu mol/L, 46 mu mol/L, 92 mu mol/L, 184 mu mol/L or 367 mu mol/L when the cono double alpha-helix antibacterial peptide with the amino acid sequence shown in SEQ ID NO. 3 is selected as the antibacterial peptide for preparing the Pichia pastoris resistant product.
14. The use of the cono double alpha-helix antibacterial peptide according to claim 9 for preparing antifungal products, wherein the concentration of the antibacterial peptide is 183 or 366 mu mol/L when the cono double alpha-helix antibacterial peptide with the amino acid sequence shown as SEQ ID NO. 4 is selected from the antibacterial agent, the medicine and the additive for preparing the Pichia pastoris resistant products.
15. The use of the cono double alpha-helix antibacterial peptide according to claim 10, wherein the concentration of the antibacterial peptide is 97.5 mu mol/L, 195 mu mol/L, 390 mu mol/L or 780 mu mol/L when the cono double alpha-helix antibacterial peptide with the amino acid sequence shown as SEQ ID NO. 1 is selected from the antibacterial agent, the medicament and the additive for preparing the product for resisting escherichia coli.
16. The use of the cono double alpha helix antibacterial peptide according to claim 10, wherein the concentration of the antibacterial peptide is 17.9 mu mol/L, 35.8 mu mol/L, 71.5 mu mol/L, 143 mu mol/L, 286 mu mol/L or 572 mu mol/L when the cono double alpha helix antibacterial peptide with the amino acid sequence shown in SEQ ID NO. 2 is selected as the antibacterial peptide for preparing the product against streptococcus dysgalactiae.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211585193.2A CN115850427B (en) | 2022-12-09 | 2022-12-09 | Conus double-alpha helix antibacterial peptide, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211585193.2A CN115850427B (en) | 2022-12-09 | 2022-12-09 | Conus double-alpha helix antibacterial peptide, preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115850427A CN115850427A (en) | 2023-03-28 |
| CN115850427B true CN115850427B (en) | 2023-09-01 |
Family
ID=85671906
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211585193.2A Active CN115850427B (en) | 2022-12-09 | 2022-12-09 | Conus double-alpha helix antibacterial peptide, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115850427B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014023129A1 (en) * | 2012-08-07 | 2014-02-13 | 海南大学 | α-CONOTOXIN PEPTIDE, AND MEDICAL COMPOSITION AND PURPOSE THEREOF |
| CN109942691A (en) * | 2017-12-20 | 2019-06-28 | 海南医学院 | Conotoxin polypeptide CTx-btg01 and its preparation method and application |
| CN109942690A (en) * | 2017-12-20 | 2019-06-28 | 海南医学院 | Conotoxin polypeptide CTx-btg02 and its preparation method and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040203132A1 (en) * | 2003-02-28 | 2004-10-14 | Cognetix, Inc. | Conus protein disulfide isomerase |
-
2022
- 2022-12-09 CN CN202211585193.2A patent/CN115850427B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014023129A1 (en) * | 2012-08-07 | 2014-02-13 | 海南大学 | α-CONOTOXIN PEPTIDE, AND MEDICAL COMPOSITION AND PURPOSE THEREOF |
| CN109942691A (en) * | 2017-12-20 | 2019-06-28 | 海南医学院 | Conotoxin polypeptide CTx-btg01 and its preparation method and application |
| CN109942690A (en) * | 2017-12-20 | 2019-06-28 | 海南医学院 | Conotoxin polypeptide CTx-btg02 and its preparation method and application |
Non-Patent Citations (1)
| Title |
|---|
| High Throughput Identification of Novel Conotoxins from the Vermivorous Oak Cone Snail (Conus quercinus) by Transcriptome Sequencing;Bingmiao Gao等;Int J Mol Sci;第19卷(第12期);第1-17页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115850427A (en) | 2023-03-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mygind et al. | Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus | |
| Castro et al. | Hylin a1, the first cytolytic peptide isolated from the arboreal South American frog Hypsiboas albopunctatus (“spotted treefrog”) | |
| Lai et al. | An anionic antimicrobial peptide from toad Bombina maxima | |
| Mechkarska et al. | Antimicrobial peptides with therapeutic potential from skin secretions of the Marsabit clawed frog Xenopus borealis (Pipidae) | |
| US6638531B1 (en) | Antimicrobial peptides | |
| He et al. | Antimicrobial peptide diversity in the skin of the torrent frog, Amolops jingdongensis | |
| Xi et al. | Medusins: a new class of antimicrobial peptides from the skin secretions of phyllomedusine frogs | |
| CN102816770B (en) | Cotesia plutellae antimicrobial peptide defensin gene, antimicrobial peptide and application | |
| CN113214355B (en) | Special antifungal antibacterial peptide GL4W as well as preparation method and application thereof | |
| Neshani et al. | Extended-Spectrum antimicrobial activity of the Low cost produced Tilapia Piscidin 4 (TP4) marine antimicrobial peptide | |
| CN116813712B (en) | Antibacterial peptide W33 with alpha-helical structure and rich in Trp, and preparation method and application thereof | |
| Wu et al. | Limnonectins: a new class of antimicrobial peptides from the skin secretion of the Fujian large-headed frog (Limnonectes fujianensis) | |
| Yu et al. | Identification, eukaryotic expression and structure & function characterizations of β-defensin like homologues from Pelodiscus sinensis | |
| CN115850427B (en) | Conus double-alpha helix antibacterial peptide, preparation method and application thereof | |
| Zheng et al. | Novel family of antimicrobial peptides from the skin of Rana shuchinae | |
| CN113999297B (en) | Antibacterial peptide hrNCM and preparation method and application thereof | |
| CN112961218B (en) | Peptide with broad-spectrum bactericidal activity and preparation method and application thereof | |
| CN116024223A (en) | Conus alpha helix and beta sheet antibacterial peptide, and preparation method and application thereof | |
| CN101570760B (en) | Recombinant mouse beta-alexin 3 polypeptide, preparation and use thereof | |
| KR101508693B1 (en) | Novel antimicrobial peptide from Crassostrea gigas, and uses thereof | |
| Chen et al. | Cloning from tissue surrogates: antimicrobial peptide (esculentin) cDNAs from the defensive skin secretions of Chinese ranid frogs | |
| CN110627891B (en) | Method for concealing hemolytic toxic and side effects of bee venom hemolytic peptide | |
| Dolashki et al. | Antimicrobial compounds from the mucus of garden snail Cornu aspersa | |
| CN113214377B (en) | Antibacterial peptide AP-64 and preparation method and application thereof | |
| CN114195869B (en) | Peptide and preparation method 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 | ||
| CB03 | Change of inventor or designer information |
Inventor after: Huang Yu Inventor after: Li Ruihan Inventor after: Xu Junmin Inventor after: You Xinxin Inventor after: Shi Qiong Inventor before: Huang Yu Inventor before: Li Ruihan Inventor before: You Xinxin Inventor before: Shi Qiong Inventor before: Xu Junmin |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240118 Address after: 6th Floor, BGI Second Office, Building 11, Beishan Industrial Zone, No. 146 Beishan Road, Yantian District, Shenzhen, Guangdong 518000 Patentee after: SHENZHEN BGI MARINE Research Institute Patentee after: Guangdong Dabaihui Marine Technology Group Co.,Ltd. Address before: 516357 No.1 Youganpu, Xunliao Binhai Tourist Resort, Huidong County, Huizhou City, Guangdong Province Patentee before: Guangdong Dabaihui Marine Technology Group Co.,Ltd. |
|
| TR01 | Transfer of patent right |