CN115947814A - Antibacterial peptide cathelicidin-BF and expression and application thereof in pichia pastoris - Google Patents
Antibacterial peptide cathelicidin-BF and expression and application thereof in pichia pastoris Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides an antibacterial peptide cathelicidin-BF and expression and application thereof in pichia pastoris, wherein the sequence of the provided antibacterial peptide is SEQ ID NO. 1. The invention uses a pichia pastoris eukaryotic expression system to express and prepare the antimicrobial peptide cathelicidin-BF, and in vitro bacteriostasis tests and chicken infection model experiments prove that the antimicrobial peptide cathelicidin-BF has strong inhibition effect on staphylococcus aureus and escherichia coli in vitro and has better treatment effect on chicken staphylococcus aureus and escherichia coli, thereby providing reference for the application of the antimicrobial peptide replacing antibiotics in the production practice in the subsequent research.
Description
Technical Field
The invention belongs to the technical field of antibacterial peptide screening application, and particularly relates to an antibacterial peptide cathelicidin-BF and expression and application thereof in pichia pastoris.
Background
Due to the advantages of wide antibacterial spectrum, high antibacterial activity, high sterilization speed, low toxic and side effects and the like, the antibacterial peptide is favored in recent years and gradually becomes an anti-infection candidate. Although antimicrobial peptides are ubiquitous in the natural world, they are complicated in kind, but have problems such as low yield and difficulty in purification. At present, the main domestic method for preparing the antibacterial peptide is to obtain the antibacterial peptide by a gene engineering expression method. Common expression cells include Escherichia coli, bacillus subtilis, pichia pastoris, etc. Among them, bacillus subtilis is widely used in the preparation of antibacterial peptides due to the advantages of low endotoxin content and high expression level. And the molecular weight of the antibacterial peptide is small, and if only a recombinant plasmid of a single copy gene is constructed, the phenomenon that the expression quantity is too low or even not expressed is easy to occur.
Disclosure of Invention
The invention aims to provide an antibacterial peptide Catlicidin-BF and expression and application thereof in pichia pastoris, thereby making up the defects of the prior art.
The invention firstly provides an antibacterial peptide Catlicidin-BF; wherein the antimicrobial peptide comprises:
1) A polypeptide having the sequence KFFRKLKKSVKKRAKEFFKKPRVIGVSPF (SEQ ID NO: 1);
2) A polypeptide which is obtained by substituting, deleting and adding one or a plurality of amino groups on the sequence in the step 1) and has the function in the step 1);
a specific nucleotide sequence of the nucleic acid segment for coding the antibacterial peptide is as follows (SEQ ID NO: 2):
AAATTCTTTC GTAAGTTGAA GAAGTCCGTG AAGAAAAGAG CTAAAGAATT TTTTAAGAAG CCTAGAGTCA TTGGAGTGAG TATCCCATTT。
in still another aspect of the present invention, there is provided a tandem polypeptide comprising not less than two of said antimicrobial peptides cathelicidin-BF;
a specific nucleotide sequence of the nucleic acid fragment encoding the tandem polypeptide is as follows (SEQ ID NO: 3):
AAATTTTTTAGAAAGTTGAAAAAGTCTGTAAAGAAAAGAGCTAAAGAATTCTTTAAGAAGCCAAGAGTCATTGGTGTTAGTATTCCTTTCGGAACCGGTGATCCTAAGTTCTTCAGAAAGCTTAAAAAATCTGTAAAAAAGAGAGCAAAAGAATTTTTCAAAAAACCACGAGTTATCGGAGTTTCAATACCATTCGGTACGGGAGATCCTAAGTTCTTTAGAAAATTGAAAAAATCCGTAAAGAAGAGAGCTAAGGAGTTTTTTAAAAAGCCTAGAGTTATAGGTGTTTCTATCCCATTC。
the antibacterial peptide or the tandem polypeptide provided by the invention is used for preparing antibacterial products;
the antimicrobial peptide or the tandem polypeptide of the present invention can also be used as a feed additive.
The invention also provides a method for recombinant expression of the antibacterial peptide or the tandem polypeptide, which is to recombinant express the polypeptide in pichia pastoris.
The invention also provides a recombinant pichia pastoris, wherein the recombinant pichia pastoris carries a nucleic acid fragment for coding the bacterial peptide or the tandem polypeptide.
The invention uses a pichia pastoris eukaryotic expression system to express and prepare the antimicrobial peptide cathelicidin-BF, and in vitro bacteriostasis tests and chicken infection model experiments prove that the antimicrobial peptide cathelicidin-BF has strong inhibition effect on staphylococcus aureus and escherichia coli in vitro and has better treatment effect on chicken staphylococcus aureus and escherichia coli, thereby providing reference for the application of the antimicrobial peptide replacing antibiotics in the production practice in the subsequent research.
Drawings
FIG. 1: and (3) inducing the obtained antimicrobial peptide cathelicidin-BF SDS-PAGE protein gel electrophoresis identification picture, wherein M:180kD Marker;1: (ii) an uninduced empty vector yeast supernatant; 2: induced empty vector yeast supernatant; 3: inducing recombinant vector yeast supernatant;
FIG. 2: a WB identification chart for inducing the obtained antimicrobial peptide Catlicidin-BF, wherein M is 180kD Marker;1 inducing empty carrier yeast supernatant; 2: yeast supernatant of the induced recombinant vector;
FIG. 3: the acidified composite protein Tricine SDS page gel electrophoresis identification picture, M is 180kD Marker;1-3:20% composite protein after acetic acid hydrolysis;
FIG. 4 is a schematic view of: an in vitro bacteriostatic test chart of a staphylococcus aureus standard strain, wherein 1:10 μ L ampicillin (25 μ g/mL) solution; 2:10 μ L amoxicillin (25 μ g/mL) solution; 3: 10. Mu.L of streptomycin (25. Mu.g/mL) solution; 4:10 μ L (35 μ g/mL) of large intestine group antimicrobial peptide; 5:10 μ L (30 μ g/mL) of zymogen antimicrobial peptide;
FIG. 5: in vitro bacteriostatic test chart of escherichia coli drug-resistant strains, wherein 1:10 μ L ampicillin (25 μ g/mL) solution; 2:10 μ L (29 μ g/mL) of yeast expressing the antimicrobial peptide Catlicidin-BF; 3:10 μ L (25 μ g/mL) of amoxicillin solution; 4:10 μ L (35 μ g/mL) of antimicrobial peptide Catlicidin-BF expressed in E.coli;
FIG. 6: an in vitro bacteriostatic test chart of a staphylococcus aureus drug-resistant strain, wherein 1:10 μ L ampicillin (25 μ g/mL) solution; 2:10 μ L (25 μ g/mL) of amoxicillin solution; 3:10 μ L (35 μ g/mL) of Cathlicidin-BF in large intestine group; 4:10 μ L (32 μ g/mL) of the yeast group Cathlicidin-BF.
Detailed Description
The information of the reagent consumables and the strain plasmids used in the examples of the present invention are as follows: pichia cells were purchased from Saimer Feishale science (China) Co., ltd and vector plasmid pPICZ α -A was purchased from Sigma Aldrich (Shanghai) trade Co., ltd. Xho I restriction enzyme, xba I restriction enzyme, sacI restriction enzyme and EcoRI restriction enzyme, as well as T4DNA ligase, rTaq enzyme and Ex Taq enzyme were purchased from Baori physician technology (Beijing) Co., ltd. The DNA gel recovery kit is a product of Beijing Tiangen Biochemical technology Co., ltd; zeocin is available from Invitrogen Biotech Inc. of America; biotin, D-glucose, peptone-Y, D-sorbitol, yeast extract, etc. are available from Thermo corporation, USA under the brand OXOID; reagents such as methanol, trichloroacetic acid, acetone, and PBS buffer were purchased from Qingdao Haibo Biotech, inc.
The specific components of the culture medium are as follows:
1. the low-salt liquid LB culture medium comprises the following components in percentage by weight:
the low-salt LB solid culture medium is prepared by adding 2% agar powder into the above components, and Zeocin TM Instead, 100. Mu.g/mL.
2. The YPD liquid culture medium comprises the following components in percentage by weight:
tryptone (tryptone) 20g
Yeast extract (yeast extract) 10g
900mL of double distilled water
100mL of glucose;
the preparation method comprises autoclaving at 120 deg.C for 15min, and adding glucose.
3. The YPDSZ + liquid culture medium comprises the following components in percentage by weight:
4. the BMGY culture medium comprises the following components in percentage by weight:
the present invention will be described in detail below with reference to examples and the accompanying drawings.
Example 1: construction of recombinant expression vector for recombinant expression of antimicrobial peptide Catlicidin-BF
The antimicrobial peptide Cathalidin-BF having the sequence KFFRKLKKSVKKRAKEFFKKPRVIGVCSPF (SEQ ID NO: 1) is screened from chicken bursa of Fabricius.
Three cathelicidin-BF were connected in series by linker (GTGDP), and the gene sequence (SEQ ID NO: 3) was synthesized by Biotechnology engineering (Shanghai) GmbH, and PCR amplification was performed after the synthesis of the target gene.
The synthesized gene is subjected to double enzyme digestion by virtue of Xho I restriction enzyme and Xba I restriction enzyme, and simultaneously the vector plasmid pPICZ alpha-A is subjected to double enzyme digestion, and the target gene of the antibacterial peptide is inserted into the expression plasmid pPICZ alpha-A. The successfully identified positive plasmid was named pPICZ alpha-A-BF and sequenced to confirm that the correct fragment was inserted.
Taking a proper amount of pichia pastoris cells stored in an ultra-low temperature refrigerator, inoculating the pichia pastoris cells into a YPD solid plate culture medium without antibiotics by a three-section streaking method, and placing the inoculated YPD solid plate culture medium in an (inverted) constant temperature incubator under the culture conditions of 30 ℃ and 72 hours.
Picking single colony (preferably containing satellite colony) in the third streak area, inoculating in a proper amount (3-5 ml) of liquid YPD culture medium, and placing in a constant temperature shaking incubator under the culture conditions: the culture was carried out overnight at 30 ℃ and 220 r/min. Taking 0.5mL of overnight-cultured bacterium liquid, inoculating the bacterium liquid into 50mL of YPD liquid culture medium without antibiotics, and placing the YPD liquid culture medium in a constant-temperature shaking incubator, wherein the culture conditions are as follows: cultured overnight at 30 ℃ and 220 r/min. When the concentration of the bacterial liquid reaches OD of more than 1.2 600 Below 1.5, preparation of competent cells is started. And (3) centrifuging the bacterium solution meeting the conditions to collect thalli, wherein the centrifugation conditions are as follows: 4 ℃, 2500r/min and 5min. Resuspending the cells with appropriate amount of precooled double distilled water (sterilization), taking care of gentle operation, centrifugally collecting the cells after resuspension, wherein the centrifugal conditions are as follows: 4 ℃, 1500r/min and 5min. Take 10mL of precooled 1M D-sorbitol (sterilized) to resuspend the cells, take care of gentle handling, and after resuspension, centrifuge again to collect the cells. Taking appropriate amount of precooled 1M D-sorbitol (sterilized) for resuspension of cells, taking care of gentle operation, taking care of determining OD value in the process of resuspension, and when OD is reached 600 And when the cell number is not less than 1.00, obtaining the pichia pastoris competent cell.
The recombinant vector plasmid was extracted and single digested with SacI restriction enzyme. After the completion of the digestion, 4. Mu.L of the digested product was sampled and detected by electrophoresis on 1% agarose gel. An appropriate amount of double distilled water was additionally added to the remaining product, the amount of the system was replenished to 200. Mu.L, and the reaction was carried out with equal volumes of phenol, phenol: extracting the chloroform mixture and the chloroform reagent once respectively, adding 10% by volume of 3M NaAC (pH 5.2) and 2.5 times by volume of precooled absolute ethyl alcohol after extraction, mixing the mixture evenly, placing the mixture in a low-temperature refrigerator (-20 ℃), centrifuging the mixture (12000 r/min and 5 min) after 1h, washing the precipitate for 2 times by using 70% of ethyl alcohol after centrifugation is finished, and re-suspending the precipitate to the final concentration of 1 mu g/mu L by using double distilled water (sterile) after drying.
And (3) uniformly mixing 10 mu g of recombinant plasmid subjected to single enzyme digestion with 80 mu L of competent cells (sorbitol for resuspension), adding into a 0.2cm quartz electric rotating cup (precooled in advance and soaked in 70% ethanol for half an hour), and placing the rotating cup on ice for 5min after the sample addition. The electric rotating cup (wiping off water on the cup wall) after the treatment is placed in an electric rotating instrument for electric rotation, and the conditions are as follows: 1.5kV, 200. Omega., 25. Mu.F. After the electrotransfer is completed, 1mL of 1mol/L D-sorbitol is added immediately and mixed. The shock-finished mixture was transferred to a sterile centrifuge tube and placed in a thermostatted incubator at 30 ℃ for 2h.
After the culture is finished, a proper amount of transformant is taken and inoculated into YPDS plate culture medium (the configuration method is shown in a table 3.4) containing bleomycin, after the inoculation is finished, the plate is placed in a constant temperature incubator for culture at 30 ℃ for more than 72h, and the growth condition of the transformant is observed during the period. Taking an empty pPICZ alpha-A vector, carrying out restriction enzyme single digestion to linearize the vector, carrying out electric transformation to pichia pastoris competent cells, carrying out antibiotic identification plate screening, and taking the screened positive strain as a control in a subsequent test.
Inoculating the transformed recombinant Pichia pastoris cells into a YPDS plate culture medium containing bleomycin, selecting positive single colonies, inoculating into 2mL of a YPDS liquid culture medium containing bleomycin again for culturing (30 ℃,220 r/min) for 16h, and continuing PCR amplification by using a yeast genome in a bacterial liquid as a template after the culture is finished, wherein the primers are 5'AOX and 3' AOX which are universal yeast primers and a target gene specific primer. After the PCR was completed, 5. Mu.L PCR product was identified by 1% agarose gel electrophoresis. The result shows that the target gene of the antibacterial peptide is correctly integrated into the genome of pichia pastoris.
Inoculating the screened positive recombinant expression strain into a YPD liquid culture medium containing bleomycin, and placing the YPD liquid culture medium in a constant-temperature shaking incubator at 30 ℃ and 220r/min for overnight shaking culture. Inoculating overnight cultured bacteria solution to BMGY culture mediumIn the culture, the inoculation ratio of the bacterial liquid is 1%, and the bacterial liquid is cultured at 30 ℃ and 220r/min until the bacterial liquid OD is reached 600 After reaching 4-8, centrifugally collecting thalli, wherein the centrifugal conditions are as follows: 3000r/min and 5min. After the centrifugation is finished, the supernatant is discarded, the BMMY culture medium is used for heavy suspension, the BMMY culture medium is placed in a constant-temperature shaking incubator for culture after heavy suspension, the culture conditions are 30 ℃,220r/min, and methanol with the final concentration of 50ml/L is added every 24 h. The induction time was 96h, during which samples were taken and stored every 12 h. After the induction is finished, the mixture is centrifuged for 10min at 8000r/min and 4 ℃, and the supernatant is collected.
And (3) taking the yeast supernatant subjected to centrifugation after induction, carrying out His-tag column type purification, fully and uniformly mixing the collected supernatant containing the target protein with the filler in the purification column, and placing the mixture in a refrigerator at 4 ℃ overnight to ensure that the His-tag and the nickel column are fully combined to improve the purification efficiency. Taking out the purification column, fixing, performing percolation, washing and elution on the supernatant, and respectively collecting percolation liquid and washing liquid eluent, wherein each liquid is collected by 5 tubes of Eppendorf tubes, and each tube is 500 mu L.
After methanol induction, the expression of the antimicrobial peptide cathelicidin-BF was confirmed by gel electrophoresis as shown in FIG. 1.
Meanwhile, the obtained antibacterial peptide cathelicidin-BF is subjected to western blot test verification, and the verification result proves that the protein obtained by induction expression is the antibacterial peptide cathelicidin-BF (figure 2).
Example 2: determination of biological Activity of antimicrobial peptide Catlicidin-BF
In this example, the LB broth was purchased from Qingdao Haibo Biotech, inc.; ampicillin, streptomycin, gentamicin, amoxicillin were purchased from national pharmaceutical group chemical reagents, ltd; the disposable culture dish is purchased from Shanghai Biotechnology Ltd; agar was purchased from invitrogen, inc; staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922, E058) are from the clinical laboratory center of China Ministry of health, and Escherichia coli and Staphylococcus aureus clinical drug-resistant strains are stored in the laboratory; non-immune sanhuang chicks were purchased from experimental animal breeding, jinanpun ltd.
Because the prepared antibacterial peptide sample is a compound protein formed by connecting 3 polypeptides in series,therefore, it is necessary to cleave it, and the method used is acetic acid acidification. A 20% acetic acid solution is prepared, and a sample is added into acetic acid to be dissolved according to the proportion (precipitation: 20% acetic acid = 1) under the following dissolving conditions: observing, recording and detecting once every half hour at 65 ℃ for 2-4 h. Preparation of NaHCO 3 Filtering the solution for sterilization, and treating the sample after acidolysis with NaHCO 3 Neutralizing the pH value to 7.2, centrifuging at 12000rpm at 4 deg.C for 5min, collecting protein after acid hydrolysis, concentrating, and storing in refrigerator at-20 deg.C for use.
Subjecting the acidolysis treated sample to NaHCO 3 After the solution is neutralized to pH 7.2, a proper amount of solution is taken for protein Tricine SDS Page gel electrophoresis identification.
As shown in FIG. 3, the protein size was expected to be complex (3.64 kD), the upper, middle and lower lanes indicated the upper, middle and lower bands, the lowermost band indicated the antimicrobial peptide Catlicidin-BF which was sufficiently acid-hydrolyzed in the sample solution, the middle band indicated the antimicrobial peptide Catlicidin-BF tandem complex protein which was insufficiently acid-hydrolyzed in the sample, and the uppermost band indicated the non-acid-hydrolyzed portion of the sample.
1. Determination of minimum inhibitory concentration of antibacterial peptide
The acidolysis treated antimicrobial peptide cathelicidin-BF was diluted in multiple ratios and added to sterile 96-well plates, 10. Mu.l per well from 1 st to 11 th, and no growth control was added to the 12 th well.
Test bacterial suspensions (concentrations corresponding to 0.5 McLeod's turbidimetric standard) were prepared, diluted 1: 1000 in LB broth, 100. Mu.L of antimicrobial peptide was added to each well at final concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125. Mu.g/ml, and placed in a 37 ℃ incubator at constant temperature for observation and recording at 2h intervals.
MIC was taken as the lowest concentration of antimicrobial peptide that completely inhibited bacterial growth in wells of a 96-well plate.
Table 1: MIC value table of antibacterial peptide
The result shows that the antibacterial peptide Catlicidin-BF has good inhibition and killing effects on staphylococcus aureus and escherichia coli.
2. In vitro bacteriostasis test
Antimicrobial peptide in vitro bacteriostatic tests were performed according to standard agar well diffusion method using staphylococcus aureus (ATCC 29213) and escherichia coli (ATCC 25922, E058, drug-resistant strain) as test strains. The test strains were resuspended to OD using LB liquid Medium 600 Between 0.4 and 0.6, 200 mu L of LB liquid culture medium containing agar powder at 50 ℃ is uniformly mixed with 25ml of LB liquid culture medium containing agar powder, poured on a sterile culture dish for waiting for solidification, a sterile Oxford cup is used for punching a flat plate, the culture medium in the holes is taken out, 10 mu L of antibacterial peptide is added into each hole, and the mixture is placed in an incubator at 37 ℃ for culture and observation. The specific operation steps are as follows:
preparing a bottom-layer plate culture medium: 2% nutrient agar was prepared, 10ml was poured onto each plate, and after the agar solidified, the molten medium inoculated with the test strain was overlaid on 90mm plates containing a solid bottom layer of medium. After the upper agar is solidified, punching a hole by using an oxford cup, and adding an antibacterial peptide solution and an antibiotic solution, wherein the specific operation steps are as follows:
(1) Placing common LB agar culture medium in a conical flask, heating with an electromagnetic oven, boiling to clear state, and autoclaving with other articles to be autoclaved at 121 deg.C for 15 min.
(2) Bottom layer culture medium: when the agar culture medium (agar content is 2%) after high pressure is cooled to about 55-60 deg.C, 10mL of agar is poured into a sterile plate, and placed horizontally, and after solidification, the bottom layer is obtained
(3) Bacterium layer culture medium: 20mL of LB solid medium (agar content 0.75%) cooled to about 40 ℃ was poured into a sterile centrifuge tube, and 1mL of 10 was added to each tube 7 And (3) slightly reversing and uniformly mixing the CFU/mL escherichia coli liquid and the Staphylococcus aureus liquid, slowly pouring the mixture on the bottom layer culture medium, and solidifying to obtain the bacterial layer culture medium.
(4) 1g of prokaryotic expression antibacterial peptide Catlicidin-BF and eukaryotic expression antibacterial peptide Catlicidin-BF which are freeze-dried are accurately weighed by an analytical balance respectively in a sterilized 15mL centrifugal tube, 10mL solvent is added, and the mixture is shaken up to be completely dissolved. After dissolution, the supernatant is absorbed to double dilute the sample for 6 to 10 times.
(5) And (3) punching the bacteria layer culture medium by using the autoclaved Oxford cup, wherein the depth is the depth for penetrating the bacteria layer without damaging the bottom layer, the distance between the centers of all the holes is more than 25mm, and the distance between the centers of all the holes and the edge of the culture dish is more than 15 mm.
(6) Gently pick out the residual culture medium in the wells with sterile forceps, and add 10. Mu.l of the antibacterial peptide sample to each well by pipetting the antibacterial peptide sample into the wells.
(7) The culture dish is placed in a biochemical incubator at 37 ℃ for upright culture, and the results are observed after the culture is carried out for 16-18 h. The diameter of the zone was measured with a vernier caliper and recorded.
The test result is shown in figure 4, the diameter of the antibacterial peptide cathelicidin-BF 10 mu L solution (28 mu g/mL) expressed by pichia pastoris to the escherichia coli inhibition zone reaches 33.9mm; the diameter of the 10 mu L solution (30 mu g/mL) to the inhibition zone of staphylococcus aureus reaches 26.8mm, which is equivalent to the diameter of the inhibition zone of 10 mu L amoxicillin solution (25 mu g/mL).
3. In vitro bacteriostatic test of drug-resistant strain
As shown in fig. 5, the results of measuring the diameter of the zone of inhibition are as follows: the diameter of an inhibition zone of the ampicillin solution is 16.5mm, the diameter of a inhibition zone of the yeast expression antibacterial peptide Catlicidin-BF is 31.2mm, the diameter of an inhibition zone of the amoxicillin solution is 17.3mm, and the diameter of an inhibition zone of the large intestine expression antibacterial peptide Catlicidin-BF is 30.5mm.
The result shows that the bacteriostasis effect of the antibacterial peptide group is obviously better than that of the antibiotic group, and the difference between the inhibition of the antibacterial peptide to drug-resistant bacteria and the inhibition of the antibacterial peptide to a standard strain is small under the same dosage, which indicates that the antibacterial peptide can have better resistance to the drug resistance of bacteria.
The in vitro bacteriostatic test result of the staphylococcus aureus drug-resistant strain is shown in fig. 6, and the diameter of an ampicillin bacteriostatic circle is 9.5mm; the diameter of an amoxicillin solution bacteriostatic zone is 10.2mm; the diameter of the cathelicidin-BF inhibition zone of the large intestine group is 20.5mm; the diameter of a Cathlicidin-BF inhibition zone of the yeast group is 27.5mm.
The results show that the cathelicidin-BF antibacterial peptide has good antibacterial effect and also has obvious inhibition effect on drug-resistant bacteria.
Claims (10)
1. An antimicrobial peptide, comprising:
1) A polypeptide having the sequence of SEQ ID NO. 1;
2) A polypeptide which is obtained by substituting, deleting and adding one or a plurality of amino groups on the sequence in 1) and has the function in 1).
2. A nucleic acid fragment encoding the antimicrobial peptide of claim 1.
3. The nucleic acid fragment of claim 2, wherein the sequence of said nucleic acid fragment is SEQ ID NO 2.
4. A tandem polypeptide comprising not less than two antimicrobial peptides of claim 1.
5. A nucleic acid fragment encoding the tandem polypeptide of claim 4.
6. The nucleic acid fragment of claim 5, wherein the sequence of said nucleic acid fragment is SEQ ID NO. 3.
7. Use of the antimicrobial peptide of claim 1 or the tandem polypeptide of claim 4 in the preparation of an antimicrobial article.
8. Use of the antimicrobial peptide of claim 1 or the tandem polypeptide of claim 4 in the preparation of an antimicrobial preparation.
9. A recombinant Pichia pastoris that carries a nucleic acid fragment encoding the antibacterial peptide of claim 1 or the tandem polypeptide of claim 4.
10. A method for preparing the antibacterial peptide of claim 1 or the tandem polypeptide of claim 4, wherein the method is prepared by using the recombinant Pichia pastoris of claim 9.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101412753A (en) * | 2008-09-27 | 2009-04-22 | 中国科学院昆明动物研究所 | Bungarus fasciatus antibacterial peptide cathelicidin-BF, and genes and uses thereof |
| WO2017186942A1 (en) * | 2016-04-28 | 2017-11-02 | Lysando Ag | Antimicrobial agents against salmonella bacteria |
| CN109395060A (en) * | 2018-11-05 | 2019-03-01 | 苏州大学 | A kind of application of inherent immunity activating peptide |
| CN115209909A (en) * | 2020-01-02 | 2022-10-18 | 伊迪生物治疗公司 | Delivery compositions and methods |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101412753A (en) * | 2008-09-27 | 2009-04-22 | 中国科学院昆明动物研究所 | Bungarus fasciatus antibacterial peptide cathelicidin-BF, and genes and uses thereof |
| WO2017186942A1 (en) * | 2016-04-28 | 2017-11-02 | Lysando Ag | Antimicrobial agents against salmonella bacteria |
| CN109395060A (en) * | 2018-11-05 | 2019-03-01 | 苏州大学 | A kind of application of inherent immunity activating peptide |
| CN115209909A (en) * | 2020-01-02 | 2022-10-18 | 伊迪生物治疗公司 | Delivery compositions and methods |
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| Title |
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