CN115850427B - Conus double-alpha helix antibacterial peptide, preparation method and application thereof - Google Patents
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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.
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| CN109942691A (en) * | 2017-12-20 | 2019-06-28 | 海南医学院 | Conotoxin polypeptide CTx-btg01 and its preparation method and application |
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