A DAMP4-DiPGLa-H fusion protein DD1, its preparation method and application

Abstract

This invention discloses a DAMP4-DiPGLa-H fusion protein DD1, its preparation method, and its applications. DAMP4 and DiPGLa-H are fused using the TEV protease recognition sequence ENLYFQG and the flexible-to-rigid linker sequence GPGS. The corresponding fusion protein DD1 is then expressed intracellularly via a recombinant expression vector. Cells are lysed by heating under high salt conditions, and purified by centrifugation and salting out after cooling. The purified fusion protein DD1 is cleaved using TEV protease, and purified by isoelectric precipitation to obtain the target recombinant protein G-DiPGLa-H. The antibacterial activity of G-DiPGLa-H is then verified.

Description

Technical Field

[0001] This invention relates to the field of agricultural science and technology, specifically to the construction of recombinant vectors and recombinant bacteria based on the DAMP4-DiPGLa-H fusion protein gene sequence, and the biosynthesis and antibacterial activity testing of the DAMP4-DiPGLa-H fusion protein and the DiPGLa-H recombinant protein. Background Technology

[0002] Antimicrobial peptides (AMPs) possess excellent antibacterial activity and are an effective, green, and safe alternative to antibiotics. PGLa, an antimicrobial peptide derived from the skin secretions of the African clawed frog, exhibits antibacterial effects against both bacteria and fungi. Tomislav Rončević et al. designed artificially modified peptides with enhanced antibacterial activity, such as DiPGLa-H, by truncating AMPs and constructing tandem repeat sequences. DiPGLa-H consists of 20 amino acid residues with the amino acid sequence KIAKVALKALKIAKVALKAL, exhibiting a typical α-helix structure and being readily soluble in water. Furthermore, DiPGLa-H demonstrates low cytotoxicity and hemolytic activity, showing great application potential.

[0003] The chemical synthesis of DiPGLa-H is complex and expensive. Utilizing genetic engineering techniques to heterologously express and biosynthesize antimicrobial peptides in microbial systems is an important direction for the application of antimicrobial peptides. However, DiPGLa-H exhibits antibacterial activity against commonly used expression hosts, and no research has yet achieved its biosynthesis. Furthermore, biosynthesized antimicrobial peptides suffer from complex purification processes and low yields. Traditional purification processes often use affinity-tagged proteins such as GST, His, and SUMO, which require expensive chromatographic purification methods after use to produce antimicrobial peptides, hindering large-scale production. To address this, CN117164722A proposed expressing the DAMP4-FR-FO fusion protein D2L using the DAMP4 tag, but it did not conduct in vivo experiments on the antimicrobial effect of the fusion protein D2L using animal models, nor did it investigate the effect of the fusion protein D2L on drug-resistant bacteria, thus affecting its practical application.

[0004] In addition, the fusion protein expressed in "Refolding and Purification Method of Mycomycin MP1106 Fusion Protein" is an inclusion body in the cell, which needs to be refolded before being cleaved by TEV protease. Furthermore, no antibacterial experiment was performed on the obtained MP1106 recombinant protein; only the functional analysis of the fusion protein was conducted from the perspective of the higher-order structure of the protein. Summary of the Invention

[0005] The purpose of this invention is to provide a DAMP4-DiPGLa-H fusion protein DD1, its preparation method, and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In one aspect, a DAMP4-DiPGLa-H fusion protein is provided, wherein the amino acid sequence of the fusion protein is obtained by fusing DAMP4 with DiPGLa-H through a tag linking sequence (e.g., a flexible-to-rigid linking sequence GPGS) and a protease recognition sequence as a cleavage site (e.g., the TEV protease recognition sequence ENLYFQG).

[0008] Preferably, the amino acid sequence of the fusion protein is as shown in SEQ.ID.NO.2 (i.e., the amino acid sequence of the fusion protein DD1).

[0009] Secondly, a recombinant antimicrobial peptide is provided, which is a DiPGLa-H recombinant protein formed by cleaving the aforementioned DAMP4-DiPGLa-H fusion protein with a protease recognition sequence at its amino terminus. For example, the antimicrobial peptide is the target recombinant protein (amino acid sequence: GKIAKVALKALKIAKVALKAL; denoted as G-DiPGLa-H) obtained by cleaving the corresponding product of the fusion protein DD1 after expression by a host bacterium with TEV protease, i.e., the biosynthesized G-DiPGLa-H, wherein the cleaved TEV protease recognition sequence is a glycine (G) residue linked to the amino terminus of DiPGLa-H.

[0010] Thirdly, a gene sequence is provided that encodes the aforementioned DAMP4-DiPGLa-H fusion protein, namely the gene sequence of the DAMP4-DiPGLa-H fusion protein.

[0011] Fourthly, a recombinant expression vector is provided, which contains the gene sequence of the aforementioned DAMP4-DiPGLa-H fusion protein. The vector is constructed by inserting the gene sequence into an Escherichia coli expression vector such as pET28a (which contains elements such as a lactose operator that can induce expression using the inserted target gene sequence), for example, pET28a-DD1.

[0012] Fifthly, a recombinant bacterium is provided, which is a host bacterium containing the aforementioned recombinant expression vector. For example, the recombinant bacterium is an engineered Escherichia coli strain that uses a vector clone obtained by introducing the recombinant expression vector pET28a-DD1 into Escherichia coli and screening it, or an engineered Escherichia coli strain that serves as a prokaryotic cell expression system for the fusion protein DD1.

[0013] Sixthly, a method for preparing a DAMP4-DiPGLa-H fusion protein is provided, comprising the following steps:

[0014] Step 1: Based on the amino acid sequences of DAMP4 and DiPGLa-H, design the amino acid sequence of the DAMP4-DiPGLa-H fusion protein and the corresponding target gene sequence (i.e., the gene sequence of the DAMP4-DiPGLa-H fusion protein), and synthesize the designed target gene sequence; the amino acid sequence of the fusion protein (e.g., the amino acid sequence of the fusion protein DD1) is obtained by fusing DAMP4, the linker sequence (i.e., the above-mentioned tag linker sequence), the protease recognition sequence (i.e., the above-mentioned protease recognition sequence as a cleavage site), and DiPGLa-H in sequence from the amino terminus to the carboxyl terminus.

[0015] Step 2: Construct a recombinant expression vector containing the gene sequence of the DAMP4-DiPGLa-H fusion protein (specifically, connect the target gene sequence to the expression vector backbone to obtain a recombinant expression vector); transform the recombinant expression vector (e.g., pET28a-DD1) into a host bacterium to obtain a genetically engineered bacterium for expressing the DAMP4-DiPGLa-H fusion protein (e.g., fusion protein DD1);

[0016] Step 3: The DAMP4-DiPGLa-H fusion protein was induced to be expressed intracellularly using the genetically engineered bacteria. The expression product was then separated and purified to obtain the DAMP4-DiPGLa-H fusion protein (e.g., fusion protein DD1).

[0017] Preferably, the amino acid sequence of the fusion protein is as shown in SEQ.ID.NO.2 (i.e., the amino acid sequence of the fusion protein DD1).

[0018] Preferably, the gene sequence of the fusion protein is shown in SEQ.ID.NO.1 (i.e., the gene sequence of the fusion protein DD1), and the nucleotide sequence is designed by codon preference optimization of E. coli.

[0019] Preferably, the host bacterium is selected from Escherichia coli BL21(DE3).

[0020] Preferably, the separation and purification specifically includes the following steps: after inducing expression with the corresponding Escherichia coli genetically engineered bacteria, the cells are lysed by heating and under high salt conditions (0.6~0.9 M sodium sulfate) (60~90℃, 20~40min), cooled, and purified by centrifugation to remove impurities and salting out (adjusting the pH to 2.5~3.5 with HCl or H3PO4, then diluting and allowing to stand); the purified DAMP4-DiPGLa-H fusion protein (e.g., fusion protein DD1) is redispersed for later use.

[0021] Seventhly, the gene sequence of the DAMP4-DiPGLa-H fusion protein, the recombinant expression vector, and the application of the recombinant bacteria in the production (biosynthesis) of antimicrobial peptides are provided. For example, a method for preparing recombinant antimicrobial peptides is specifically provided, including the following steps:

[0022] The DAMP4-DiPGLa-H fusion protein prepared above was cleaved using a protease (for fusion protein DD1, a TEV protease with a recognition sequence compatible with the TEV protease it was fused with was used, and the cleavage was carried out under the conditions of 30~60 U / mL and ≤50 min) to release the antimicrobial peptide (for fusion protein DD1, the released protein is the DiPGLa-H recombinant protein with the cleaved TEV protease recognition sequence at the amino terminus, i.e., the above-mentioned G-DiPGLa-H), and then isoelectric precipitation was performed (pH=6.8, to precipitate "DAMP4-GPGS-ENLYFQ") to obtain the recombinant antimicrobial peptide.

[0023] Eighthly, the use of the aforementioned recombinant antimicrobial peptide in the preparation of a medicament for treating infectious diseases caused by Gram-negative bacteria, Gram-positive bacteria, or their drug-resistant strains is provided.

[0024] Preferably, the amino acid sequence of the recombinant antimicrobial peptide is: GKIAKVALKALKIAKVALKAL; the drug-resistant strain is selected from methicillin-resistant Staphylococcus aureus, such as Mu50.

[0025] The beneficial effects of this invention are reflected in:

[0026] This invention utilizes the heat and salt tolerance of DAMP4 and designs a DiPGLa-H protein fused with DAMP4 using a protease recognition sequence (e.g., the TEV protease recognition sequence) and a linker sequence, namely the DAMP-DiPGLa-H fusion protein. After expression, this fusion protein can be used to obtain a recombinant DiPGLa-H protein with a cleaved protease recognition sequence. Taking the obtained biosynthesized G-DiPGLa-H as an example, it exhibits broad-spectrum antibacterial activity and good safety, and has potential application value, particularly in the preparation of drugs for treating drug-resistant bacterial infections.

[0027] This invention utilizes DAMP4, its linker sequence, and its protease recognition sequence to express the DAMP4-DiPGLa-H fusion protein. The fusion protein is then purified by protease cleavage to obtain antimicrobial peptides (such as the aforementioned G-DiPGLa-H). This not only solves the problem that biosynthesized antimicrobial peptides are toxic to the host and affect expression efficiency, but also allows the fusion protein to be directly separated and purified after prokaryotic induction (and is a non-chromatographic purification process), avoiding cumbersome and expensive production processes.

[0028] Furthermore, this invention rapidly induces the expression of the fusion protein DD1 (containing pET28a-DD1) in E. coli (IPTG 0.1~1 mM, cultured at 16~20°C for 16~24 h) (the amino acid sequence of the fusion protein DD1 is obtained by fusing DAMP4, a flexible-rigid linker sequence GPGS, a TEV protease recognition sequence ENLYFQG, and DiPGLa-H arranged sequentially from the amino terminus to the carboxyl terminus). After centrifuging to collect a large amount of bacterial cell precipitate, the bacterial cells are lysed and the impurities are denatured under simple heating and high-salt conditions. The impurities are removed by centrifugation and then salted out to obtain the purified fusion protein DD1. Subsequently, high-purity antimicrobial peptides (specifically referring to the aforementioned G-DiPGLa-H) are obtained through enzymatic hydrolysis (i.e., cleavage of the fusion protein DD1 using the cleavage activity of the TEV protease) and isoelectric precipitation. Attached Figure Description

[0029] Figure 1 This is a plasmid map of the recombinant expression vector pET28a-DD1.

[0030] Figure 2 These are PCR identification results. Lanes 1-5 in Figure A are examples of transformant screening results for pET28a-DD1 transformed into DH5α, and lanes 1-5 in Figure B are examples of transformant screening results for pET28a-DD1 transformed into BL21(DE3).

[0031] Figure 3 This is a Tricine-SDS-PAGE small molecule protein gel assay for the induced expression of bacterial cells, the purified fusion protein DD1 (TEV protease digestion for 0 min), and the products of TEV protease digestion for 10-50 min; in the figure, M is the marker, -IPTG is uninduced, and +IPTG is after IPTG induction.

[0032] Figure 4 This is a graph showing the supernatant (containing G-DiPGLa-H) and precipitate products after isoelectric precipitation purification, as detected by Tricine-SDS-PAGE electrophoresis; M in the graph is the marker.

[0033] Figure 5 These are the HPLC and MS chromatograms of purified G-DiPGLa-H.

[0034] Figure 6 It is G-DiPGLa-H and DiPGLa-H pair E. coli Graph showing the determination of the minimum inhibitory concentration (MIC) of K88 (with fusion protein DD1 as the control).

[0035] Figure 7This is a graph showing the hemolytic activity of purified G-DiPGLa-H.

[0036] Figure 8 This is a graph showing the survival rate (A) and organ-borne colony count (B) of mice infected with drug-resistant Staphylococcus aureus Mu50 in the intraperitoneal cavity, after treatment with PBS, fusion protein DD1, G-DiPGLa-H, and DiPGLa-H. Detailed Implementation

[0037] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the invention.

[0038] (I) Gene sequence design and synthesis of DAMP4-DiPGLa-H fusion protein

[0039] DAMP4 and the antimicrobial peptide DiPGLa-H were linked together using the linker sequence GPGS and the TEV protease recognition sequence ENLYFQG (the TEV protease cleaves this sequence between Q and G) to form DAMP4-GPGS-ENLYFQG-DiPGLa-H (or DAMP4-GPGS-TEV-DiPGLa-H), which is the fusion protein DD1 (amino acid sequence shown in Table 1-1). The gene sequence of the fusion protein DD1 was optimized according to the codon preference of E. coli, and Nco I and Xho I restriction sites were added before and after the optimized gene sequence (see Table 1-2), respectively. The gene sequence with restriction sites was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0040] Table 1-1. Amino acid sequence of fusion protein DD1

[0041]

[0042] Table 1-2. Gene sequence of fusion protein DD1

[0043]

[0044] Preliminary experiments show that fusing DAMP4 with DiPGLa-H using the following amino acid sequence—the TEV protease recognition sequence ENLYFQG and the moderately flexible / rigid linker sequence GPGS—can prevent the toxicity of DiPGLa-H to the host *E. coli*. Furthermore, the combined effects of high salt and high temperature lead to cell lysis and protein denaturation. The heat- and high-salt resistance of DAMP4 ensures the stability of DAMP4-DiPGLa-H (specifically, the fusion protein DD1) at 90°C and in 0.8M sodium sulfate, while other contaminating proteins precipitate. This provides a material basis for obtaining high-purity recombinant DiPGLa-H protein (amino acid sequences shown in Tables 1-3) by enzymatically hydrolyzing the fusion protein DD1 with the TEV protease.

[0045] Table 1-3. Amino acid sequence of the recombinant DiPGLa-H protein released by enzymatic decomposition.

[0046]

[0047] Note: The amino acid residues marked with an underline in Table 1-3 (specifically "G") are represented by the letter G in the name of the target recombinant protein listed in the "Name" column, "G-DiPGLa-H"; the remaining 20 amino acid residues "KIAKVALKALKIAKVALKAL" are the amino acid sequence of the antimicrobial peptide DiPGLa-H.

[0048] (II) Construction of recombinant expression vectors

[0049] The construction process and results of the recombinant expression vector for inserting the target gene sequence into pET28a are as follows: Figure 1 As shown, the empty plasmid pET28a (Sangon Biotech (Shanghai) Co., Ltd., B540183) was double-digested with Nco I and Xho I, and the linearized vector fragment was obtained by gel recovery. The synthesized gene fragment (containing the target gene sequence) was amplified and recovered, and after double digestion, it was ligated to the linearized vector fragment using T4 DNA ligase and transformed into DH5α (DL1001S, Shanghai Weidi Biotechnology Co., Ltd.). The selected single clones were verified by colony PCR. Figure 2 A) and sequencing. Correct single clones were selected and cultured in LB medium, plasmid (i.e., pET28a-DD1) was extracted and transformed into the expression host BL21(DE3) (EC1002S, Shanghai Weidi Biotechnology Co., Ltd.). The selected single clones were then verified by colony PCR. Figure 2 B) Sequencing was performed, and the correct single clones were preserved and used for expression experiments. The primers for colony PCR identification are shown in Table 2.

[0050] Table 2. Colony PCR Primers

[0051]

[0052] (III) Induction and purification of fusion protein DD1

[0053] Preserved *E. coli* BL21 (DE3) carrying the pET28a-DD1 plasmid was streaked onto LB agar plates and incubated overnight at 37°C. Single colonies were picked and inoculated into 10 mL LB medium containing 50 µg / mL kanamycin and incubated overnight at 37°C with continuous shaking. Then, the culture was inoculated at a 1% ratio into 500 mL LB medium containing kanamycin and incubated at 37°C until an OD600 of 0.6–0.8 was reached. IPTG was added to a final concentration of 1 mM, and the mixture was incubated at 16°C with shaking for 16 hours. The bacterial pellet was collected by centrifuging the resulting fermentation broth at 12000 g for 10 minutes at room temperature, and the supernatant was discarded.

[0054] The bacterial precipitate was resuspended in 50 mL of equilibration buffer (50 mM sodium chloride and 25 mM disodium hydrogen phosphate, pH 7.5). Sodium sulfate was added to a final concentration of 0.8 M, and the mixture was incubated in a 90°C water bath for 30 minutes, then cooled to room temperature. The mixture was centrifuged at 39,000 g for 10 minutes at 4°C, and the supernatant was collected. This step removed the contaminating proteins, including the fusion protein DD1, from the precipitate. The pH of the supernatant was then adjusted to 3 with hydrochloric acid, diluted five-fold with ultrapure water, and allowed to stand at room temperature for 30 minutes. Under this low pH environment, the fusion protein DD1 precipitated. The precipitate was then centrifuged at 39,000 g for 10 minutes at 4°C, and the collected precipitate was resuspended in 10 mL of ultrapure water to obtain a suspension of the fusion protein DD1.

[0055] The induced and purified fusion protein DD1 was analyzed by Tricine-SDS-PAGE small molecule protein gel analysis, and the results are as follows: Figure 3 As shown, after IPTG induction, the band of the target fusion protein (i.e., fusion protein DD1) darkened near 14.4 kDa, and the purified fusion protein DD1 band was also at this location with a higher concentration. The yield of fusion protein DD1 was determined to be approximately 3.5 g / L.

[0056] (iv) Enzymatic hydrolysis and purification of G-DiPGLa-H

[0057] 400 U of TEV protease was added to the DD1 fusion protein suspension, followed by incubation at 30°C for 50 min, with 10-min intervals for sampling. The samples were then analyzed using Tricine-SDS-PAGE small molecule protein gel chromatography. Figure 3As shown, with increasing enzymatic hydrolysis time, the concentration of the fusion protein DD1 decreased due to cleavage, releasing DAMP4-GPGS-ENLYFQ and G-DiPGLa-H (the latter having a lower molecular weight and not shown in the results). Figure 3 The fusion protein DD1 was completely cleaved at 50 min.

[0058] After 50 min of enzymatic digestion, 10 mL of the sample was adjusted to pH 6.8 with NaOH and allowed to stand at room temperature for 30 min. The sample was then centrifuged at 38000g for 20 min at 4℃. The supernatant and precipitate were collected separately and labeled. Tricine-SDS-PAGE electrophoresis was performed for detection. Figure 4 As shown, the 11.6 kDa DAMP4-GPGS-ENLYFQ protein band was mainly detected in the precipitate, while the 2.6 kDa protein band detected in the supernatant was the target recombinant protein G-DiPGLa-H.

[0059] The supernatant was analyzed by RP-HPLC and LC-MS, and the results are as follows: Figure 5 As shown, HPLC results indicated that G-DiPGLa-H exhibited a main peak at 8.4 minutes, with a protein purity of approximately 96%. MS results revealed the consistency between the theoretical molecular weight (2607.11 Da) and the actual molecular weight (2620.40 Da) of G-DiPGLa-H. Ultimately, 31.1 mg of G-DiPGLa-H could be purified from 1 L of fermentation broth, achieving a yield of 31.1 mg / L.

[0060] (v) Antibacterial and bactericidal activities of G-DiPGLa-H

[0061] Escherichia coli (K88, ATCC25922), Pseudomonas aeruginosa (ATCC27853), Acinetobacter baumannii (ATCC19606), and Staphylococcus aureus (ATCC29213, Mu50) were removed from a -80℃ freezer, thawed on ice, streaked, and incubated overnight. Single colonies were inoculated into 3 mL of LB or TSB medium and incubated overnight (12-16 h) on a shaker at 37℃. The overnight bacterial culture was corrected using MHB medium to a McFarland turbidity standard concentration of 0.5 (1-2 × 10⁻⁶). 8The bacterial suspension was then diluted 1:100 with MHB medium to correct the concentration (CFU / mL). A sterile 96-well plate was used. The drug (DiPGLa-H, G-DiPGLa-H, or fusion protein DD1) was serially diluted and added 100 μL to wells 1-8, and 100 μL of bacterial suspension to wells 1-9. Wells containing only MHB medium served as a blank control. The plates were incubated at 37°C for 24 hours. Turbidity was observed in each well, and the OD600 value was measured using a microplate reader to determine the minimum inhibitory concentration (MIC). 4 μL of the concentration above the MIC was selected from each well and spotted onto solid MH medium. The plates were incubated overnight at 37°C. Viable bacteria were counted, and the concentration that caused 99.9%–99.99% bacterial kill in these wells was taken as the minimum bactericidal concentration (MBC). Each bacterial strain was repeated in triplicate, with three replicates.

[0062] The difference in antibacterial activity between DiPGLa-H and G-DiPGLa-H was compared by determining the minimum inhibitory concentration (MIC). The results are as follows: Figure 6 As shown, the minimum inhibitory concentration (MIC) of both the chemically synthesized antimicrobial peptide DiPGLa-H and the purified G-DiPGLa-H against Escherichia coli K88 was 8 μg / mL. Meanwhile, the fusion protein DD1 did not exhibit any antimicrobial activity.

[0063] In addition, the results of the antibacterial and bactericidal activity assays (MIC and MBC determinations) of G-DiPGLa-H against other Gram-negative and Gram-positive bacteria are shown in Table 3.

[0064] Table 3. MIC (MBC) of G-DiPGLa-H and DiPGLa-H against bacteria: unit (µg / mL)

[0065]

[0066] The results showed that G-DiPGLa-H had an effect on A. baumannii ATCC19606 exhibited the best antibacterial and bactericidal activity (MIC / MBC = 2 µg / mL) against Gram-positive bacteria. S. aureus ATCC29213 showed good inhibitory effects (MIC=8µg / mL) against drug-resistant bacteria. S. aureus Mu50 exhibits significantly stronger antibacterial activity than common non-drug-resistant bacteria (specifically referring to...). S. aureus (ATCC29213), and G-DiPGLa-H also showed significantly stronger antibacterial and bactericidal activity against Mu50 than DiPGLa-H; overall, the biosynthesized G-DiPGLa-H exhibited broad-spectrum antibacterial and bactericidal activity against the tested Gram-negative and Gram-positive bacteria (including drug-resistant bacteria).

[0067] (vi) Assessment of the hemolytic activity of G-DiPGLa-H

[0068] A 20 mL fresh blood sample was drawn from a healthy piglet using a sterile syringe and centrifuged at 1000 g for 5 min. The erythrocyte pellet was aspirated and resuspended in PBS (pH 7.4) to obtain a 1% (v / v) erythrocyte dilution. Equal volumes of the erythrocyte dilution and different concentrations (1–128 μg / mL) of G-DiPGLa-H were mixed in 96-well plates and incubated at 37°C for 1 hour. The mixture was centrifuged at 1000 g for 10 min, and the supernatant was transferred to a new 96-well plate. The OD value was measured at 576 nm using a microplate reader to assess hemoglobin release. This experiment was repeated three times, with each experiment performed in triplicate. The results of the G-DiPGLa-H hemolytic activity test are as follows: Figure 7 As shown, with the increase of G-DiPGLa-H concentration, G-DiPGLa-H only showed a low hemolytic ability (5.1%, 7.2%) in the range of 64~128 μg / mL. However, this concentration is much lower than the maximum values ​​of MIC and MBC of G-DiPGLa-H against different strains determined in (V). Therefore, biosynthesized G-DiPGLa-H has good in vivo application value.

[0069] (VII) Application of G-DiPGLa-H in a mouse model of intraperitoneal infection

[0070] Six- to eight-week-old male C57BL / 6 mice were selected as experimental subjects and divided into eight groups: a survival group and a dissection group injected with PBS; a survival group and a dissection group injected with the fusion protein DD1; a survival group and a dissection group injected with DiPGLa-H; and a survival group and a dissection group injected with G-DiPGLa-H. Each survival group contained 16 mice, and each dissection group contained 5 mice. At the start of the experiment, all eight groups of mice were intraperitoneally injected with a lethal dose of Staphylococcus aureus Mu50. Thirty minutes later, two groups were injected with G-DiPGLa-H at a concentration of 32 μg / mL. Injections were repeated every 6 hours for a total of three times. Two groups were injected with DiPGLa-H and the fusion protein DD1 in the same manner. The other two groups were injected with PBS as controls. All mice in the dissection group were sacrificed on the second day after infection. The heart, liver, spleen, and kidneys were removed from the dissected mice, homogenized, and diluted with PBS to 10⁻⁶. 7 The serially diluted buffer (100 μL) was spread onto LBA plates and incubated overnight at 37°C. Viability and bacterial counts were analyzed using GraphPad Prism software. The experimental protocol and animal use were approved by the Animal Protection and Use Committee of Northwest A&F University.

[0071] The experimental results showed that all mice in the PBS and DD1 fusion protein survival groups infected with drug-resistant Staphylococcus aureus Mu50 died within 5 days, while the survival rate of mice in the G-DiPGLa-H and DiPGLa-H survival groups increased to 50% after 7 days of treatment. Figure 8 A). Bacterial load assays in mouse organs revealed that the bacterial load in all organs was significantly reduced after treatment in both the G-DiPGLa-H and DiPGLa-H dissection groups. Specifically, the bacterial load in the heart, spleen, liver, and kidneys of mice in the G-DiPGLa-H dissection group was reduced by 98.69%, 99.45%, 99.90%, and 99.92%, respectively, compared to the PBS dissection group. Figure 8 B). By Figure 8 B also shows that the bacterial load in the liver and spleen of mice in the G-DiPGLa-H dissection group was significantly lower than that in the DiPGLa-H dissection group. These results demonstrate that biosynthesized G-DiPGLa-H can effectively treat intraperitoneal infection in mice caused by drug-resistant Mu50, and has promising potential as a novel antimicrobial peptide.

Claims

1. A DAMP4-DiPGLa-H fusion protein, characterized in that: The fusion protein is composed of DAMP4 and DiPGLa-H fused through a tag linking sequence and a protease recognition sequence serving as a cleavage site. The amino acid sequence of the fusion protein is shown in SEQ.ID.NO.

2.

2. A recombinant antimicrobial peptide, characterized in that: The antimicrobial peptide is a DiPGLa-H recombinant protein with a cleaved protease recognition sequence at its amino terminus, and the amino acid sequence of the antimicrobial peptide is shown in SEQ.ID.NO.

3.

3. A recombinant expression vector, characterized in that: The vector contains a gene sequence encoding the DAMP4-DiPGLa-H fusion protein as described in claim 1.

4. A recombinant bacterium, characterized in that: The recombinant bacteria contains the recombinant expression vector as described in claim 3.

5. A method for preparing a DAMP4-DiPGLa-H fusion protein, characterized in that: Includes the following steps: Step 1: Based on the amino acid sequence of the DAMP4-DiPGLa-H fusion protein, design and synthesize the corresponding target gene sequence. The fusion protein is obtained by fusing DAMP4, the linker sequence, the protease recognition sequence, and DiPGLa-H sequentially from the amino terminus to the carboxyl terminus. The amino acid sequence of the fusion protein is shown in SEQ.ID.NO.

2. Step 2: The target gene sequence is linked to the expression vector backbone to obtain a recombinant expression vector. The recombinant expression vector is then transformed into a host bacterium to obtain a genetically engineered bacterium for expressing the DAMP4-DiPGLa-H fusion protein. Step 3: The DAMP4-DiPGLa-H fusion protein was induced to be expressed intracellularly using the genetically engineered bacteria. The expression product was then separated and purified to obtain the DAMP4-DiPGLa-H fusion protein.

6. A method for preparing the recombinant antimicrobial peptide as described in claim 2, characterized in that: Includes the following steps: The DAMP4-DiPGLa-H fusion protein was prepared using the method described in claim 5; the DAMP4-DiPGLa-H fusion protein was cleaved using a protease that is compatible with the recognition sequence of the fused protease, and then the recombinant DiPGLa-H protein, i.e., the recombinant antimicrobial peptide, was obtained by isoelectric precipitation.

7. The method for preparing the recombinant antimicrobial peptide according to claim 6, characterized in that: The fusion protein was cleaved using the TEV protease.

8. The use of the recombinant antimicrobial peptide as described in claim 2 in the preparation of a medicament for treating infectious diseases caused by Gram-negative or Gram-positive bacteria, characterized in that: The Gram-negative bacteria are Escherichia coli, Acinetobacter baumannii, or Pseudomonas aeruginosa, and the Gram-positive bacteria are Staphylococcus aureus.

9. The application according to claim 8, characterized in that: The Staphylococcus aureus was selected from methicillin-resistant Staphylococcus aureus.

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