Barnacle chitin antibacterial peptide, engineering algal strain of barnacle chitin antibacterial peptide as well as preparation method and application of engineering algal strain

Abstract

The invention relates to the field of gene engineering, and particularly discloses a barnacle chitin antibacterial peptide, an engineering algal strain of the barnacle chitin antibacterial peptide and a preparation method and application of the engineering algal strain. Wherein the amino acid sequence of the barnacle chitin antibacterial peptide is as shown in SEQ ID NO. 1; or an amino acid sequence which is obtained by substitution and / or deletion and / or addition of one or more amino acids of SEQ ID NO.1 and has the same function as SEQ ID NO.1; or an amino acid sequence which has 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the SEQ ID NO.1 and has the same function as the SEQ ID NO.1. The brand-new barnacle chitin antibacterial peptide obtained in the application has a good antibacterial effect, bacteria can be killed through a cell protection barrier, and the cell hemolysis rate at the minimum inhibition concentration is 7.2%.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to a barnacle chitin antimicrobial peptide, an engineered algae strain of a barnacle chitin antimicrobial peptide, and a preparation method and application thereof. Background Art

[0002] Antimicrobial peptides (or host defense peptides) are a class of naturally occurring polypeptides that are an important component of the natural immune defense of most organisms against pathogens. Antimicrobial peptides have been found in animals, plants, microorganisms, fungi, and bacteria, and are generally described as low molecular weight, highly cationic molecules with broad-spectrum activity against different pathogens including Gram-negative bacteria, Gram-positive bacteria, protozoa, fungi, or viruses. Most antimicrobial peptides are composed of less than 100 amino acids and are small oligopeptides. They exert their antimicrobial effects through common mechanisms, such as destroying and penetrating the bacterial membrane of pathogens. As a potential alternative to traditional antibiotics, antimicrobial peptides are attractive because of their unique antimicrobial mechanisms, and some antimicrobial peptides are currently undergoing clinical trials.

[0003] Although some antimicrobial peptides have been shown to have therapeutic effects on systemic infections, there are currently no commercial antimicrobial peptides on the market. The main constraints facing the commercial production of such peptides include high production costs, insufficient in vivo stability, and possible toxicity to the host.

[0004] Therefore, the prior art needs to be improved. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present application is to provide a barnacle chitin antimicrobial peptide, aiming to solve the problems of high production cost, low yield, poor stability and possible toxicity to the host of antimicrobial peptides.

[0006] The technical solution of this application is as follows:

[0007] In a first aspect of the present application, a barnacle chitin antimicrobial peptide is provided, wherein the amino acid sequence of the barnacle chitin antimicrobial peptide is:

[0008] (a1) SEQ ID NO. 1; or

[0009] (a2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO.1 and which has the same function as the amino acids in the sequence shown in SEQ ID NO.1; or

[0010] (a3) an amino acid sequence that has 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO.1 and has the same function as the amino acids in the sequence shown in SEQ ID NO.1.

[0011] In a second aspect of the present application, a nucleic acid is provided, wherein the nucleic acid encodes the barnacle chitin antimicrobial peptide according to the first aspect of the present application.

[0012] Preferably, the nucleic acid has the sequence shown in SEQ ID NO.2.

[0013] The third aspect of the present application provides a vector, wherein the vector comprises the nucleic acid of the second aspect of the present application.

[0014] The fourth aspect of the present application provides an engineered algae strain of barnacle chitin antimicrobial peptide, wherein the engineered algae strain of barnacle chitin antimicrobial peptide comprises the vector of the third aspect of the present application; the engineered algae strain of barnacle chitin antimicrobial peptide is Phaeodactylumtricornutum GES84, classified and named Phaeodactylumtricornutum, and the preservation unit is Guangdong Microbiological Culture Collection Center, the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the preservation number is GDMCC No: 64910, and the preservation date is July 24, 2024.

[0015] The fifth aspect of the present application provides a method for preparing an engineered algae strain of barnacle chitin antimicrobial peptide according to the fourth aspect of the present application, comprising the steps of: connecting the nucleic acid to the pPha-T1 expression plasmid to obtain the vector; transforming the vector into algae cells to obtain the engineered algae strain of barnacle chitin antimicrobial peptide.

[0016] Preferably, the step of transforming the vector into algae cells comprises the steps of: linearizing the vector by ScaI enzyme to obtain a linearized vector; and transforming the linearized vector into the algae cells by gene gun bombardment.

[0017] Preferably, the algae cells include Phaeodactylum tricornutum.

[0018] The sixth aspect of the present application provides an engineered algae strain of the barnacle chitin antimicrobial peptide of the first aspect of the present application and the barnacle chitin antimicrobial peptide of the fourth aspect of the present application for use in the preparation of an antimicrobial product.

[0019] Preferably, the antimicrobial product comprises at least one of drugs, feed, feed additives, preservatives, daily chemicals, fabrics, paper products, medical materials, and cell culture media.

[0020] Beneficial effects of this application:

[0021] (1) The barnacle chitin antimicrobial peptide in the present application has a good antibacterial effect and can kill bacteria through the cell protection barrier. The cell hemolysis rate at the minimum inhibitory concentration is 7.2%.

[0022] (2) The barnacle chitin antimicrobial peptide in this application uses a single-cell engineered algae strain as a bioreactor, which has stable expression, reduces production fluctuations caused by environmental changes and other factors, and is conducive to large-scale mass production; it does not require complex culture equipment and conditions, thereby reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for describing the embodiments.

[0024] Figure 1 A diagram of the expression vector of the antimicrobial peptide AaCrus5-A provided in the examples of this application;

[0025] Figure 2 This is an observation diagram of the effect of the antimicrobial peptide AaCrus5-A provided in the examples of the present application on bacterial morphology and cell membrane integrity;

[0026] Figure 3 This is an observation diagram of the engineered algae strain producing the antimicrobial peptide AaCrus5-A provided in the examples of the present application;

[0027] Figure 4 This is a Western Blot verification diagram of the antimicrobial peptide AaCrus5-A provided in the examples of the present application;

[0028] Figure 5 This is the LC-MS verification diagram of the antimicrobial peptide AaCrus5-A provided in the examples of the present application;

[0029] Figure 6 This is a graph showing the results of the hemolytic activity assay of the antimicrobial peptide AaCrus5-A provided in the examples of the present application. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings and embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present application. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0031] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance and implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the fact that ordinary technicians in the field can implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0032] Since the 21st century, due to the progress of the pharmaceutical industry and the abuse of antibiotics, antibiotic resistance has long become a medical problem faced by countries around the world, and has been listed by the World Health Organization as one of the major public health threats facing mankind in the 21st century. In this regard, the World Health Organization and global medical workers all agree that it is necessary to mobilize the resources of countries and governments around the world to solve this problem.

[0033] Antimicrobial peptides (or host defense peptides) are a class of naturally occurring polypeptides that are an important component of the natural immune defense of most organisms against pathogens. Most antimicrobial peptides are composed of less than 100 amino acids and are small oligopeptides. They exert their antimicrobial effects through common mechanisms, such as destroying and penetrating the bacterial membrane of pathogens. As a potential alternative to traditional antibiotics, antimicrobial peptides are attractive because of their unique antimicrobial mechanisms, and some antimicrobial peptides are currently undergoing clinical trials.

[0034] Although some antimicrobial peptides have been shown to have therapeutic effects on systemic infections, there are currently no commercial antimicrobial peptides on the market. The main constraints facing the commercial production of such peptides include high production costs, insufficient in vivo stability, and possible toxicity to the host.

[0035] Based on this, the first aspect of the embodiment of the present application provides a barnacle chitin antimicrobial peptide, the amino acid sequence of which is:

[0036] (a1) SEQ ID NO. 1; or

[0037] (a2) an amino acid sequence in which one or more amino acids are substituted and / or deleted and / or added to SEQ ID NO.1 and which has the same function as the amino acids in the sequence shown in SEQ ID NO.1; or

[0038] (a3) an amino acid sequence that has 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO.1 and has the same function as the amino acids in the sequence shown in SEQ ID NO.1.

[0039] Barnacles are small barnacles with colorful stripes on their shells. They are commonly found in ports around the world and attach to the surfaces of organisms and ships. The development of barnacles is influenced by microbial communities that can promote or prevent the settlement and colonization of barnacle larvae. The amazing fecundity of barnacles in a harsh and pathogen-filled environment suggests that they may have a strong innate immune system, which may include antimicrobial peptides (AMPs) with significant antimicrobial properties.

[0040] The barnacle chitin antimicrobial peptide of the first aspect of the embodiment of the present application belongs to the Crustin class of antimicrobial peptides, which is obtained from the genome of barnacles using the hidden Markov model of the Crustin class of antimicrobial peptides and is named AaCrus5-V. The barnacle chitin antimicrobial peptide is obtained by chemical synthesis or exogenous expression. After determining its minimum inhibitory concentration by the two-fold dilution method and its cytotoxicity by the hemolysis test, it was found that the barnacle chitin antimicrobial peptide of the first aspect of the embodiment of the present application has a good antibacterial effect and can kill bacteria through the cell protection barrier. The cell hemolysis rate at the minimum inhibitory concentration is 7.2%.

[0041] According to a second aspect of the embodiments of the present application, a nucleic acid is provided, which encodes the barnacle chitin antimicrobial peptide according to the first aspect of the embodiments of the present application.

[0042] Nucleic acid is an oligomer or polymer of any length, composed of nucleotides (deoxyribonucleotides and / or ribonucleotides). Nucleic acids can contain purine and / or pyrimidine bases and / or other natural (e.g., xanthine, inosine, hypoxanthine), chemically or biochemically modified (e.g., methylated), non-natural or derived nucleotide bases. In some embodiments, the sequence of the nucleic acid is shown in SEQ ID NO.2.

[0043] The third aspect of the embodiment of the present application provides a vector, which includes the nucleic acid of the second aspect of the embodiment of the present application. A vector refers to a nucleic acid carrier into which a nucleotide can be inserted. When the vector can express the protein encoded by the inserted nucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by methods such as transformation, transduction or transfection, and then the genetic material it carries is expressed in the host cell. The vector may contain a variety of control expression elements, including but not limited to promoter sequences, transcription start sequences, enhancer sequences, selection elements, reporter genes and replication start sites. Recombinant expression vectors include but are not limited to vectors for proliferation or for expression or for proliferation and expression.

[0044] In the fourth aspect of the embodiment of the present application, there is provided an engineered algae strain of a barnacle chitin antimicrobial peptide, which includes the vector of the third aspect of the embodiment of the present application. The engineered algae strain of the barnacle chitin antimicrobial peptide is Phaeodactylumtricornutum GES84, which is classified and named Phaeodactylumtricornutum, and the deposit unit is Guangdong Microbiological Culture Collection Center, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64910, and the deposit date is July 24, 2024.

[0045] According to a fifth aspect of the present application, there is provided a method for preparing an engineered algae strain of a barnacle chitin antimicrobial peptide according to the fourth aspect of the present application, comprising the steps of:

[0046] Connecting the nucleic acid of the second aspect of the present embodiment to the pPha-T1 expression plasmid to obtain a vector;

[0047] The vector is transformed into algae cells to obtain an engineered algae strain of barnacle chitin antimicrobial peptide.

[0048] In some embodiments, in some embodiments, the expression plasmid is selected from one of pPha-T1, pBYR2e, psi103, and pCHI.

[0049] In some embodiments, the expression vector of the barnacle chitin antimicrobial peptide, pPha-T1-AaCrus5-V, such as Figure 1 shown.

[0050] In some embodiments, the specific steps of transforming the vector into algae cells include: linearizing the vector by ScaI enzyme to obtain a linearized vector; and transforming the linearized vector into the algae cells by gene gun bombardment.

[0051] In some embodiments, the algae cells may be selected from seawater algae or freshwater algae, preferably, the algae cells are selected from Phaeodactylum tricornutum.

[0052] In a sixth aspect of the embodiments of the present application, a conjugate is provided, which includes a modified portion and the barnacle chitin antimicrobial peptide of the first aspect of the embodiments of the present application.

[0053] Conjugates generally refer to a combination of one type of molecule and another type of molecule formed by covalent or non-covalent bonds. In some embodiments, the modified portion of the conjugate is located at the N-terminus and / or C-terminus of the barnacle chitin antimicrobial peptide, and the modified portion includes at least one of a chemical modification, a targeting portion, a fluorescent dye, and a protein tag. For example, chemical modifications include acetylation, amination, methylation, phosphorylation, glycosylation, ubiquitination, etc.; the targeting portion encapsulates ligands, receptors, antibodies, etc.; fluorescent dyes include fluorescein isothiocyanate (FITC), etc.; protein tags include His, Flag, MBP, GFP, etc.

[0054] In the seventh aspect of the embodiments of the present application, a fusion protein is provided, which includes other polypeptides and the barnacle chitin antimicrobial peptide of the first aspect of the embodiments of the present application.

[0055] The fusion protein is the expression product of two genes recombined by DNA recombination technology. In some embodiments, the other polypeptide is located at the N-terminus and / or C-terminus of the barnacle chitin antimicrobial peptide, wherein the polypeptide can be connected to the barnacle chitin antimicrobial peptide via a linker.

[0056] In an eighth aspect of the embodiments of the present application, a composition is provided, comprising the barnacle chitin antimicrobial peptide according to the first aspect of the embodiments of the present application.

[0057] In some embodiments, the composition further comprises other active antimicrobial ingredients. The bacteria in other active antimicrobial ingredients include at least one of bacteria and fungi. For example, the bacteria in other active antimicrobial ingredients can be Staphylococcus aureus, Bacillus T2, Aeromonas hydrophila, Acinetobacter L32, Vibrio anguillarum, Vibrio parahaemolyticus, Vibrio harveyi, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, etc. Preferably, other active antimicrobial ingredients can be penicillin antibiotics, cephalosporin antibiotics, tetracycline antibiotics, etc.

[0058] In the ninth aspect of the embodiments of the present application, a barnacle chitin antimicrobial peptide according to the first aspect of the embodiments of the present application, an engineered algae strain of the barnacle chitin antimicrobial peptide according to the fourth aspect of the embodiments of the present application, a conjugate according to the sixth aspect of the embodiments of the present application, a fusion protein according to the seventh aspect of the embodiments of the present application, and a composition according to the eighth aspect of the embodiments of the present application are provided for use in the preparation of antibacterial products.

[0059] In some embodiments, the antimicrobial product comprises at least one of drugs, feed, feed additives, preservatives, daily chemicals, fabrics, paper products, medical materials, and cell culture media.

[0060] Preferably, the medicine includes at least one of an injection, an oral agent, and an external agent.

[0061] Preferably, the daily chemical products include at least one of facial cleanser, hand soap, shower gel, mouthwash, toothpaste, soap, cosmetics, feminine care lotion, laundry soap, laundry liquid, laundry powder, detergent, disinfectant, and toilet cleaning liquid.

[0062] Preferably, the paper product includes at least one of a sanitary napkin, a sanitary pad, a diaper, and a urine pad.

[0063] Preferably, the medical material includes at least one of a medical hydrogel material, a medical antibacterial material, a tissue regeneration material, and a tissue repair material.

[0064] The following is further described by specific examples. It should be noted that the experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The materials, reagents and instruments used in the examples, if specifically stated, can all be obtained from commercial sources.

[0065] Example 1 Mining of barnacle chitin antimicrobial peptide encoding genes and construction of expression vectors

[0066] Using the hidden Markov model of Crustin antimicrobial peptides, the amino acid sequence (as shown in SEQ ID NO.1) and the encoding nucleic acid sequence (as shown in SEQ ID NO.2) of the barnacle chitin antimicrobial peptide were screened from the NCBI barnacle genome; the nucleic acid sequence of the signal peptide was removed from the nucleic acid sequence, optimized according to the codon preference of Phaeodactylum tricornutum, and EcoR I and BamH I restriction enzyme sites were added to both ends of the sequence. General Biotech provided the pPha-T1 expression vector (containing the fcpA promoter), and the barnacle chitin antimicrobial peptide (AaCrus5-V) gene was synthesized by chemical synthesis, and the synthesized DNA was connected to the pPha-T1 vector through the restriction enzyme sites. The mature peptide of AaCrus5-V without the signal sequence was fused to the C-terminus of the 6×His tag to obtain the vector pPha-T1-AaCrus5-V (as shown in Figure 1 As shown), obtain the plasmid lyophilized powder.

[0067] Example 2 Transformation and screening of engineered algae strains

[0068] (1) The triangular brown algae grew to the logarithmic growth stage (density of about 7×10 6 cell·mL -1 ), harvest the algal cells by centrifugation at 1500 g for 10 min at 4°C, and wash three times with 375 mM sorbitol (4°C, sterile) to remove the medium components. Resuspend the algal cells in 100 μL of 375 mM sorbitol to a final density of 2 × 109 cell·mL -1 Take 2 mL of concentrated algae solution in the clean bench and spread it evenly in the center of antibiotic-free F / 2 solid culture medium (about 3 cm in diameter) with a spreading stick, and dry it until the algae solution can no longer flow.

[0069] (2) The recombinant plasmid was obtained from the recombinant E. coli using a plasmid extraction kit. The plasmid DNA was linearized by ScaI enzyme (1 μg DNA was added with 1 μL enzyme) and then the linearized plasmid was recovered using a kit. The linearized recombinant plasmid was concentrated to 1 μg / μL using a vacuum heating rotary evaporator. -1 .

[0070] (3) The plasmid DNA was coated in 0.5 μm gold powder through a series of steps. 50 μL of 3 mg gold powder stored in 50% glycerol was added to a 1.5 mL microcentrifuge tube and vortexed vigorously for 5 min. 6 μL of linearized plasmid DNA (1 μg / μL) was slowly added dropwise during the vigorous shaking. -1 ), 50 μL calcium chloride (2.5 M) and 20 μL spermidine (0.1 M), continue to shake for 3 minutes and then stand at room temperature for 1 minute, centrifuge for 2 seconds and discard the supernatant, add 140 μL 70% ethanol and discard the supernatant, add 140 μL anhydrous ethanol (99.7%) and discard the supernatant, add 50 μL anhydrous ethanol (99.7%), tap one side of the centrifuge tube wall and vortex at low speed for 3 seconds to resuspend the wrapped gold powder.

[0071] Before bombardment, the carrier membrane and the barrier net were sterilized at 121°C for 20 minutes in a high pressure steam sterilizer and then dried. The remaining particle bombardment instrument accessories were sterilized by irradiation with ultraviolet light for 30 minutes in a clean bench. 10 μL of resuspended gold powder was evenly spread on the middle of the carrier membrane (about 1 cm in diameter) and dried. The broken membrane (1550 psi) was soaked in 70% isopropanol for 1 minute. The bombardment device was assembled according to the instructions of the gene gun equipment manufacturer. The bombardment distance was 9 cm, and each plate containing triangular phytoplankton was bombarded twice.

[0072] (4) After the bombardment, the plates were placed in a constant temperature incubator at 20°C in the dark for 24 hours, and then the triangular algae on each non-antibiotic plate were transferred to non-antibiotic F / 2 liquid culture medium, centrifuged at 2000g for 5 minutes, and the supernatant was removed. After resuspending in 1 mL of non-antibiotic F / 2 liquid culture medium, the cells were spread on four zeocin-resistant F / 2 solid culture medium plates using a spreader stick. The cells on the plates were statically cultured in a constant temperature and light incubator for 1 to 2 months. The culture conditions were as follows: light-dark cycle ratio of 12:12h, temperature of (20.0±1)°C, and light intensity of 4000Lux.

[0073] (5) Pick a moderately sized, round, brown algae colony (e.g. Figure 2 ) and transferred to a zeocin (100 μg mL -1 ) in F / 2 liquid medium, and wild-type (WT) triangular phaeophyceae (as negative control) was transferred to F / 2 liquid medium without antibiotics and cultured to the logarithmic growth phase. Triangular phaeophyceae cells were collected by centrifugation at 8000g for 10 min. After the ceramic mortar, pestle and related equipment were treated to remove RNase, the algal cells were placed in the mortar for liquid nitrogen grinding, and RNA was extracted according to the instructions of the RNA extraction kit. The RNA concentration was determined using Nanodrop and the integrity and contamination of RNA were detected by 1.2% agarose gel electrophoresis.

[0074] RNA was reverse transcribed into complementary DNA (cDNA) using a reverse transcription kit, and cDNA diluted 100 times with ultrapure water was used as a template. PCR amplification of pPha-F / R was performed using specific primers, and 2% agarose gel electrophoresis was performed. After the electrophoresis, the agarose gel was placed under a gel imager to observe whether there were bands of the corresponding molecular weight after primer amplification. The PCR products with bands were sent to the company (Sangon) for sequencing. The spliced ​​sequencing results and the vector sequence were compared using NCBI. If the comparison results matched 100%, it meant that the transformants were successfully obtained.

[0075] The present application example uses genetic engineering technology to successfully integrate the gene sequence of barnacle chitin antimicrobial peptide into the genome of algae, and constructs an engineered algae strain that can stably express the antimicrobial peptide. This engineered algae strain has the advantages of fast growth, low cultivation cost, and environmental friendliness, laying the foundation for the large-scale production of barnacle chitin antimicrobial peptide.

[0076] Example 3 Cultivation of engineered algae strains and expression and identification of barnacle chitin antimicrobial peptides

[0077] (1) According to 6.36 g·L -1 Sodium carbonate, 10.94 g·L -1 Recipe of dithiothreitol and 12% sucrose The corresponding amount of reagents was weighed and dissolved in ultrapure water as a cell lysis solution, which was stored at -20°C, and phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 1 mM before use.

[0078] (2) The positive monoclonal algae strain obtained in Example 2 was transferred to F / 2 liquid culture medium, and the cells were ventilated in a constant temperature and light incubator (sterile air flow rate was 0.5 L min -1The following parameters were set: light-dark cycle ratio of 12:12h, temperature of (20.0±1)℃, and light intensity of 4000Lux. Figure 3 shown.

[0079] (3) When the triangular brown algae is cultivated to the logarithmic growth phase (density of about 7×10 6 cell·mL -1 ). Collect the algal cells by centrifugation at 8000 g for 10 min. Add cell lysis buffer containing 1 mM PMSF and resuspend the cells to a concentration of 5 × 10 7 cell·mL -1 , the cells were broken by ultrasound under ice bath conditions (power 240W, working 2s, stopping 3s, working 50min). After the ultrasonic treatment, the mixed solution was centrifuged (4°C, 14000g, 20min) and the supernatant was collected. After filtering twice with a 0.22μm filter membrane, nickel affinity chromatography was used to purify the target protein. The whole process was carried out in an ice box. The purified sample was placed in a dialysis bag (molecular weight cutoff less than 14kDa) and dialyzed in an equilibrium buffer (50mM tromethamine, 200mM sodium chloride, pH7.0) for 24h. The dialysis process was carried out in a 4°C cold storage.

[0080] (4) After dialysis, the lyophilized powder was dissolved in 1× PBS buffer (pH 7.4), the concentration of the purified protein was detected by a protein content determination kit, and Western Blot and LC-MS were used for identification. Figure 4 and Figure 5 shown. Figure 4 M indicates protein marker, WT indicates wild algae protein (negative control group), 1 and 2 indicate purified proteins. Figure 4 It can be seen that the size of the target protein matches that of the barnacle chitin antimicrobial peptide, indicating that the barnacle chitin antimicrobial peptide was successfully secreted and expressed. Figure 5 It can be seen that the LC-MS results match the amino acid sequence of the barnacle chitin antimicrobial peptide by 94%, which also shows that the barnacle chitin antimicrobial peptide is successfully secreted and expressed.

[0081] Through the efficient and low-cost barnacle chitin antimicrobial peptide extraction and purification technology in the embodiments of the present application, barnacle chitin antimicrobial peptides can be efficiently extracted from the culture medium of the engineered algae strain by using steps such as cell disruption, centrifugation, filtration, and chromatographic separation, and impurities can be removed through multiple purification steps to obtain high-purity barnacle chitin antimicrobial peptides.

[0082] Example 4 Determination of the antibacterial effect of barnacle chitin antimicrobial peptides

[0083] The purified, dialyzed and freeze-dried protein powder and BSA in Example 3 were dissolved in 1×PBS (pH 7.4), and the protein sample concentration was determined using a Cossinolide protein content kit. The concentration of the recombinant protein sample was adjusted to 2-128 μM, and the BSA concentration was 128 μM. 100 μL of test bacterial solution (Staphylococcus aureus, Bacillus T2, Escherichia coli, Aeromonas hydrophila, Acinetobacter L32, Vibrio anguillarum, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus; the concentration was 1×10 6 CFU·mL -1 ) were mixed with 100 μL of the test protein solution, and each well contained 5×10 5 CFU·mL -1 Test bacteria and test proteins at final concentrations of 1 to 64 μM, and 64 μM BSA as a negative control. The plate was incubated at 37°C for 24 h, and the OD was measured using an ELISA reader at 0 h and 24 h. 600 The MIC value was defined as no significant growth (OD 600 The lowest concentration with a change value less than 0.05). The test was performed with three biological replicates and three technical replicates. The test results are shown in Table 1.

[0084] As can be seen from Table 1, the chemically synthesized barnacle chitin antimicrobial peptides and the barnacle chitin antimicrobial peptides expressed by exogenous secretion of the engineered algae strains have certain inhibitory effects on Staphylococcus aureus, Bacillus T2, Escherichia coli, Aeromonas hydrophila, Acinetobacter L32, Vibrio anguillarum, Vibrio parahaemolyticus, Vibrio harveyi, and Vibrio alginolyticus, and the effect is significantly better than that of ampicillin. It further illustrates that the barnacle chitin antimicrobial peptides in the embodiments of the present application have antimicrobial effects on a variety of bacteria or fungi. In addition, it also illustrates that the engineered strains in the embodiments of the present application can stably express barnacle chitin antimicrobial peptides with antimicrobial effects, which, as a barnacle chitin antimicrobial peptide bioreactor, can ensure the widespread development of large-scale mass production of barnacle chitin antimicrobial peptides.

[0085] Table 1. Minimum inhibitory concentration of barnacle chitin antimicrobial peptides

[0086] Pathogenic microorganisms Chemical synthesis Recombinant expression of engineered algae Ampicillin Staphylococcus aureus 4 2 1 Bacillus T2 4 4 - Escherichia coli 4 4 100 Aeromonas hydrophila 16 8 50 Acinetobacter L32 8 4 - Vibrio anguillarum 4 2 - Vibrio parahaemolyticus 4 2 - Vibrio harveyi 2 2 - Vibrio alginolyticus 4 2 -

[0087] Example 5 Determination of hemolysis rate of barnacle chitin antimicrobial peptides

[0088] The purified, dialyzed and freeze-dried protein powder and BSA in Example 3 were dissolved in 1×PBS (pH 7.4) to the minimum inhibitory concentration. The decellulose sheep blood was washed five times with 0.85% saline (4°C, sterile) (until the supernatant was colorless and transparent), and after centrifugation at 1500g for 10 min at 4°C to obtain red blood cells, the red blood cells were suspended in 0.85% saline to a 4% diluent. 150 μL of 4% red blood cell diluent and 150 μL of test protein solution were mixed in a 1.5 mL microcentrifuge tube, and the final concentration of the recombinant protein was 4-64 μM. 1×PBS (pH 7.4) was used as a negative control (as 0% hemolysis), and the reaction containing 0.2% Triton X-100 was used as a positive control (as 100% hemolysis). After incubation at 37°C for 1 hour, the mixture was centrifuged at 1500g for 1 minute, 200 μL of the supernatant was transferred to a 96-well plate, and the OD was detected with an enzyme marker. 530 This experiment was repeated three times biologically and three times technically. The hemolysis rate of the sample was calculated as follows:

[0089] Hemolysis rate (%) = (A-A0) / (A 100 -A0)×100%

[0090] Where A is the absorbance of the sample at a wavelength of 570 nm;

[0091] A0, A 100 These are the absorbance values ​​of the samples treated with 1×PBS (pH 7.4) and 0.2% Triton X-100.

[0092] Test results such as Figure 6 As shown, from Figure 6 It can be seen that the hemolysis rate of barnacle chitin antimicrobial peptides was 7.2% at the minimum inhibitory concentration.

[0093] In summary, the barnacle chitin antimicrobial peptide in the present application has a good antibacterial effect, can kill bacteria through the cell protection barrier, and the cell hemolysis rate at the minimum inhibitory concentration is 7.2%. The barnacle chitin antimicrobial peptide in the present application uses a single-cell engineered algae strain as a bioreactor, and the expression is stable, which reduces the yield fluctuation caused by factors such as environmental changes, and is conducive to large-scale mass production; it does not require complex culture equipment and conditions, and reduces production costs.

[0094] It should be understood that the application of the present application is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to this application.

Claims

1. A barnacle chitin antimicrobial peptide, characterized in that: The amino acid sequence of the barnacle chitin antimicrobial peptide is: (a1) SEQ ID NO. 1; or (a2) an amino acid sequence in which one or more amino acids in SEQ ID NO.1 are substituted and / or deleted and / or added and which has the same function as the amino acids in the sequence shown in SEQ ID NO.1; or (a3) an amino acid sequence that has 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO.1 and has the same function as the amino acids in the sequence shown in SEQ ID NO.

1.

2. A nucleic acid, characterized in that The nucleic acid encodes the barnacle chitin antimicrobial peptide according to claim 1.

3. The nucleic acid according to claim 2, characterized in that The nucleic acid has the sequence shown in SEQ ID NO.

2.

4. A carrier, characterized in that The vector comprises the nucleic acid according to claim 2 or 3.

5. An engineered algae strain of barnacle chitin antimicrobial peptide, characterized in that: The engineered algae strain of the barnacle chitin antimicrobial peptide comprises the vector according to claim 4; The engineered algae strain of the barnacle chitin antimicrobial peptide is Phaeodactylumtricornutum GES84, classified and named Phaeodactylumtricornutum, and the preservation unit is Guangdong Microbial Culture Collection Center, the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the preservation number is GDMCC No: 64910, and the preservation date is July 24, 2024.

6. A method for preparing the engineered algae strain of barnacle chitin antimicrobial peptide according to claim 5, characterized in that: Includes steps: Connecting the nucleic acid to the pPha-T1 expression plasmid to obtain the vector; The vector is transformed into algae cells to obtain the engineered algae strain of the barnacle chitin antimicrobial peptide.

7. The preparation method according to claim 6, characterized in that: The method of transforming the vector into algae cells comprises the steps of: The vector is linearized by ScaI enzyme to obtain a linearized vector; The linearized vector is transformed into the algae cells by gene gun bombardment.

8. The preparation method according to claim 6, characterized in that: The algae cells include Phaeophytum tricornutum.

9. Use of the barnacle chitin antimicrobial peptide according to claim 1 or the engineered algae strain of the barnacle chitin antimicrobial peptide according to claim 5 in the preparation of antimicrobial products.

10. The use according to claim 9, characterized in that: The antibacterial product comprises at least one of medicines, feeds, feed additives, preservatives, daily chemicals, fabrics, paper products, medical materials, and cell culture media.

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