DAMP4-DiPGLa-H fusion protein DD1 as well as preparation method and application thereof

Abstract

The invention discloses a DAMP4-DiPGLa-H fusion protein DD1 as well as a preparation method and application of the DAMP4-DiPGLa-H fusion protein DD1. DAMP4 and DiPGLa-H are fused through a TEV protease recognition sequence ENLYFQG and a connecting sequence GPGS with moderate flexibility and rigidity, and a corresponding fusion protein DD1 is expressed in cells through a recombinant expression vector; heating and cracking cells under the action of high salt, cooling, and purifying through centrifugal impurity removal and salting-out; tEV protease is used for cutting the fusion protein DD1 obtained through purification, the target recombinant protein G-DiPGLa-H is obtained after isoelectric precipitation purification, and the antibacterial activity of the G-DiPGLa-H is verified.

Description

Technical Field

[0001] The present invention relates to the field of agricultural science and technology, and in particular to the construction of a recombinant vector and recombinant bacteria based on a DAMP4-DiPGLa-H fusion protein gene sequence, and the biosynthesis and antibacterial activity test of a DAMP4-DiPGLa-H fusion protein and a DiPGLa-H recombinant protein. Background Art

[0002] Antimicrobial peptides (AMPs) have good antibacterial activity and are an effective, green and safe alternative to antibiotics. PGLa is an antimicrobial peptide derived from the skin secretions of African clawed frogs. It has a certain antibacterial effect on bacteria and fungi. Tomislav Rončević et al. designed artificially modified peptides with better antibacterial activity, such as DiPGLa-H, by truncating it and constructing tandem repeat sequences. DiPGLa-H is composed of 20 amino acid residues with an amino acid sequence of KIAKVALKALKIAKVALKAL. It has a typical α-helical structure and is easily soluble in water. At the same time, DiPGLa-H has low cytotoxicity and hemolytic activity, and has great application potential.

[0003] The chemical synthesis process of DiPGLa-H is complex and expensive. Using genetic engineering technology to express biosynthetic antimicrobial peptides in microbial systems through heterologous expression is an important direction for realizing the application of antimicrobial peptides. However, DiPGLa-H has antibacterial activity on commonly used expression hosts, and no relevant research has yet been conducted to achieve the biosynthesis of DiPGLa-H. In addition, biosynthesized antimicrobial peptides have the problems of complex purification process and low yield. For example, traditional purification processes use affinity tag proteins such as GST, His, and SUMO, and these tags often require expensive chromatography purification methods to produce antimicrobial peptides after use, which hinders the large-scale production of antimicrobial peptides. For this reason, CN117164722A proposes to express DAMP4-FR-FO fusion protein D2L using DAMP4 tag, but does not use animal models to conduct in vivo experiments on the antimicrobial effect of fusion protein D2L, and does not investigate the effect of fusion protein D2L on drug-resistant bacteria, thereby affecting its practical application.

[0004] In addition, the fusion protein expressed in the "Refolding and Purification Method of Plectomycin MP1106 Fusion Protein" is an inclusion body in the cell, which needs to be renatured and then cut by TEV protease. No antibacterial experiment was performed on the prepared MP1106 recombinant protein, and only the functional analysis of the fusion protein was carried out from the perspective of the protein's higher-level structure. Summary of the invention

[0005] The purpose of the present invention is to provide a DAMP4-DiPGLa-H fusion protein DD1 and a preparation method and application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: In the first aspect, a DAMP4-DiPGLa-H fusion protein is provided, the amino acid sequence of which is obtained by fusing DAMP4 and DiPGLa-H via a tag connection sequence (e.g., a connection sequence with moderate flexibility and rigidity GPGS) and a protease recognition sequence serving as a cleavage site (e.g., a TEV protease recognition sequence ENLYFQG).

[0007] Preferably, the amino acid sequence of the fusion protein is shown in SEQ.ID.NO.2 (ie, the amino acid sequence of the fusion protein DD1).

[0008] In the second aspect, a recombinant antimicrobial peptide is provided, which is a DiPGLa-H recombinant protein formed by cleaving the above-mentioned DAMP4-DiPGLa-H fusion protein by protease, and has a protease recognition sequence after cleavage at the amino end. For example, the antimicrobial peptide is a target recombinant protein (amino acid sequence: GKIAKVALKALKIAKVALKAL; denoted as G-DiPGLa-H) obtained by cleaving the corresponding product of the fusion protein DD1 expressed by the host bacteria by TEV protease, that is, biosynthesized G-DiPGLa-H, wherein the TEV protease recognition sequence after cleavage is a glycine (G) residue connected to the amino end of DiPGLa-H.

[0009] In a third aspect, a gene sequence is provided, which is used to encode the above-mentioned DAMP4-DiPGLa-H fusion protein, that is, the gene sequence of the DAMP4-DiPGLa-H fusion protein.

[0010] In a fourth aspect, a recombinant expression vector is provided, which contains the gene sequence of the above-mentioned DAMP4-DiPGLa-H fusion protein, and the vector is constructed by inserting the gene sequence into an Escherichia coli expression vector such as pET28a (the plasmid contains elements such as a lactose operator that can utilize the inserted target gene sequence for inducible expression), such as pET28a-DD1.

[0011] In a fifth aspect, a recombinant bacterium is provided, which is a host bacterium containing the above-mentioned recombinant expression vector. For example, the recombinant bacterium is a genetically engineered E. coli bacterium for vector cloning obtained by introducing the recombinant expression vector pET28a-DD1 into E. coli and screening, or a genetically engineered E. coli bacterium for a prokaryotic cell expression system of the fusion protein DD1.

[0012] In a sixth aspect, a method for preparing a DAMP4-DiPGLa-H fusion protein is provided, comprising the following steps: Step 1: 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) according to the amino acid sequences of DAMP4 and DiPGLa-H, 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, a linker sequence (i.e., the above-mentioned tag linker sequence), a 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; Step 2: construct a recombinant expression vector containing the gene sequence of the DAMP4-DiPGLa-H fusion protein (specifically, the target gene sequence is connected to the expression vector backbone to obtain a recombinant expression vector); transform the recombinant expression vector (such as pET28a-DD1) into a host bacterium to obtain a genetically engineered bacterium for expressing the DAMP4-DiPGLa-H fusion protein (such as fusion protein DD1); Step 3: Using the genetically engineered bacteria, the DAMP4-DiPGLa-H fusion protein is induced to express in the cell, and then the expression product is separated and purified to obtain the DAMP4-DiPGLa-H fusion protein (eg, fusion protein DD1).

[0013] Preferably, the amino acid sequence of the fusion protein is shown in SEQ.ID.NO.2 (ie, the amino acid sequence of the fusion protein DD1).

[0014] Preferably, the gene sequence of the fusion protein is as shown in SEQ.ID.NO.1 (ie, the gene sequence of fusion protein DD1), and the nucleotide sequence is designed by optimizing the codon preference of Escherichia coli.

[0015] Preferably, the host bacteria is selected from Escherichia coli BL21 (DE3).

[0016] Preferably, the separation and purification specifically includes the following steps: after inducing expression by the corresponding Escherichia coli genetically engineered bacteria, the cells are lysed by heating and high salt (0.6~0.9 M sodium sulfate) (60~90°C, 20~40min), and after cooling, they are purified by centrifugal removal of impurities and salting out (adjusting the pH to 2.5~3.5 with HCl or H3PO4, then diluting and standing); the purified DAMP4-DiPGLa-H fusion protein (such as fusion protein DD1) is redispersed for later use.

[0017] In the seventh aspect, the gene sequence of the DAMP4-DiPGLa-H fusion protein, the recombinant expression vector, and the use of the recombinant bacteria in the production (biosynthesis) of antimicrobial peptides are provided. For example, a method for preparing a recombinant antimicrobial peptide is specifically provided, comprising the following steps: The DAMP4-DiPGLa-H fusion protein prepared above was cleaved by protease (for the fusion protein DD1, TEV protease compatible with the TEV protease recognition sequence to which it was fused was used, and cleavage was performed under the conditions of 30-60U / mL and enzymatic hydrolysis ≤50min) to release the antimicrobial peptide (for the fusion protein DD1, specifically 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 (pH=6.8, to precipitate "DAMP4-GPGS-ENLYFQ") was performed to obtain the recombinant antimicrobial peptide.

[0018] In an eighth aspect, provided is the use of the above-mentioned recombinant antimicrobial peptide in the preparation of a drug for treating infectious diseases caused by Gram-negative bacteria, Gram-positive bacteria or drug-resistant strains thereof.

[0019] 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.

[0020] The beneficial effects of the present invention are embodied in: The present invention utilizes the high temperature and high salt resistance of DAMP4, and utilizes a protease recognition sequence (such as a TEV protease recognition sequence) and a connection sequence to design a DiPGLa-H protein fused with DAMP4, namely a DAMP-DiPGLa-H fusion protein. After expression, the fusion protein can be used to obtain a DiPGLa-H recombinant protein with a cleaved protease recognition sequence, taking the obtained biosynthesized G-DiPGLa-H as an example, which has a broad-spectrum antibacterial activity and good safety, and has potential application value in the preparation of drugs for anti-drug resistant bacterial infection diseases.

[0021] The present invention utilizes DAMP4 and a linker sequence and a protease recognition sequence to express a DAMP4-DiPGLa-H fusion protein, and obtains an antimicrobial peptide (such as the above-mentioned G-DiPGLa-H) by cleaving the purified fusion protein with a protease, which not only solves the problem that the biosynthesized antimicrobial peptide is toxic to the host and affects the expression efficiency, but also the fusion protein can be directly separated and purified (and non-chromatographic purification) after prokaryotic induced expression, thus avoiding a cumbersome and expensive production process.

[0022] Furthermore, the present invention uses Escherichia coli (containing pET28a-DD1) to quickly induce expression (IPTG 0.1-1 mM, culture at 16-20°C for 16-24h) of a fusion protein DD1 (the amino acid sequence of the fusion protein DD1 is obtained by fusing DAMP4, a flexible and moderately rigid connection sequence GPGS, a TEV protease recognition sequence ENLYFQG and DiPGLa-H arranged in sequence from the amino terminus to the carboxyl terminus), collects a large amount of bacterial precipitates by centrifugation, lyses the bacterial cells and denatures impurities under simple heating and high-salt conditions, removes the impurity precipitates by centrifugation, and then performs salting out to obtain the purified fusion protein DD1; thereafter, high-purity antimicrobial peptides (specifically the above-mentioned G-DiPGLa-H) are obtained by enzymatic hydrolysis (i.e., utilizing the cleavage activity of the TEV protease to cleave the fusion protein DD1) and isoelectric precipitation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the plasmid map of the recombinant expression vector pET28a-DD1.

[0024] Figure 2 The following are the results of PCR identification. Lanes 1 to 5 in Figure A are examples of the results of screening transformants transformed from pET28a-DD1 into DH5α, and lanes 1 to 5 in Figure B are examples of the results of screening transformants transformed from pET28a-DD1 into BL21 (DE3).

[0025] Figure 3 This is a picture of the induced expression of bacteria detected by Tricine-SDS-PAGE small molecule protein gel, the purified fusion protein DD1 (TEV protease digestion 0min) and the product of TEV protease digestion 10-50min; in the picture, M is Marker, -IPTG is uninduced, and +IPTG is after IPTG induction.

[0026] Figure 4 This is a picture of the supernatant (containing G-DiPGLa-H) and the precipitated product after isoelectric precipitation purification detected by Tricine-SDS-PAGE electrophoresis; M in the picture is a marker.

[0027] Figure 5 It is the HPLC and MS chart of G-DiPGLa-H after purification.

[0028] Figure 6 G-DiPGLa-H and DiPGLa-H E. coli Minimum inhibitory concentration (MIC) determination of K88 (fusion protein DD1 as control).

[0029] Figure 7 This is the hemolytic activity test chart of purified G-DiPGLa-H.

[0030] Figure 8 The graph shows the effects of PBS, fusion protein DD1, G-DiPGLa-H, and DiPGLa-H on the survival rate (A) and the number of colony counts in organs (B) of mice infected with drug-resistant Staphylococcus aureus Mu50 in the mouse abdominal cavity. DETAILED DESCRIPTION

[0031] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. The embodiments are only used to explain the present invention, rather than to limit the protection scope of the present invention.

[0032] 1. Design and synthesis of the gene sequence of DAMP4-DiPGLa-H fusion protein DAMP4 and the antimicrobial peptide DiPGLa-H were connected through the linker sequence GPGS and the TEV protease recognition sequence ENLYFQG (TEV protease cuts the sequence between Q and G) to form DAMP4-GPGS-ENLYFQG-DiPGLa-H (or DAMP4-GPGS-TEV-DiPGLa-H), i.e., the fusion protein DD1 (the amino acid sequence is shown in Table 1-1). When designing the gene sequence of the fusion protein DD1, it was optimized according to the codon preference of Escherichia 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.

[0033] Table 1-1. Amino acid sequence of fusion protein DD1

[0034] Table 1-2. Gene sequence of fusion protein DD1

[0035] Preliminary experiments showed that DAMP4 was fused with DiPGLa-H through the following amino acid sequence: TEV protease recognition sequence ENLYFQG and flexible and rigid linker sequence GPGS. The introduction of DAMP4 tag will avoid the toxicity of DiPGLa-H to the host Escherichia coli. In addition, the combined effects of high salt and high temperature will lead to cell lysis and protein denaturation. The high temperature and high salt resistance of DAMP4 endows DAMP4-DiPGLa-H (specifically fusion protein DD1) to remain stable at 90°C and 0.8M sodium sulfate, and other impurities are precipitated. This provides a material basis for enzymatically digesting the fusion protein DD1 with TEV protease and obtaining a high-purity DiPGLa-H recombinant protein (amino acid sequence shown in Table 1-3).

[0036] Table 1-3. Amino acid sequence of DiPGLa-H recombinant protein released by enzymatic hydrolysis

[0037] Note: The underlined amino acid residues (specifically "G") in Tables 1-3 are represented by the letter G in the name of the target recombinant protein "G-DiPGLa-H" listed in the "Name" column; the remaining 20 amino acid residues "KIAKVALKALKIAKVALKAL" are the amino acid sequence of the antimicrobial peptide DiPGLa-H.

[0038] (II) Construction of recombinant expression vector The process and results of inserting the target gene sequence into the recombinant expression vector pET28a are shown in Figure 1 As shown, the empty plasmid pET28a (Shanghai Sangon Biotechnology Co., Ltd., B540183) was double-digested with Nco I and Xho I, and the linearized vector fragment was recovered by gel recovery; the synthesized gene fragment (including the target gene sequence) was amplified and recovered, and after the same double-digestion, it was ligated with the linearized vector fragment by T4 DNA ligase and transformed into DH5α (DL1001S, Shanghai Weidi Biotechnology Co., Ltd.), and the selected single clones were verified by colony PCR ( Figure 2 A) and sequencing. The correct single clone was picked and shaken in LB medium, the plasmid (i.e., pET28a-DD1) was extracted and transformed into the expression host BL21 (DE3) (EC1002S, Shanghai Weidi Biotechnology Co., Ltd.), and the selected single clone was verified by colony PCR ( Figure 2 B) and sequenced, and the correct single clone was preserved and used for expression experiments. The identification primers for colony PCR are shown in Table 2.

[0039] Table 2. Colony PCR primers

[0040] (III) Inducible expression and purification of fusion protein DD1 The deposited E. coli BL21 (DE3) carrying the pET28a-DD1 plasmid was streaked onto LB plates and cultured overnight at 37°C. A single colony was picked and inoculated into 10 mL LB medium containing 50 µg / mL kanamycin and cultured overnight at 37°C with continuous shaking. Then, the culture was inoculated into 500 mL LB medium containing kanamycin at a ratio of 1% and cultured at 37°C until OD600 reached 0.6~0.8. IPTG was added to a final concentration of 1 mM and cultured at 16°C with shaking for 16 hours. The bacterial precipitate was collected by centrifugation of the resulting fermentation broth at 12000 g for 10 minutes at room temperature, and the supernatant was discarded.

[0041] The bacterial precipitate was resuspended in 50 mL of equilibration buffer (50 mM sodium chloride and 25 mM sodium dihydrogen phosphate, pH 7.5). Sodium sulfate was added to a final concentration of 0.8 M and incubated in a 90°C water bath for 30 minutes, then cooled to room temperature. The mixture was centrifuged at 4°C and 39,000 g for 10 minutes, and the supernatant was collected. The supernatant obtained in this step contained fusion protein DD1 and a small amount of impurities, and the impurities in the precipitate were removed. The pH of the supernatant was then adjusted to 3 with hydrochloric acid, diluted five times with ultrapure water, and allowed to stand at room temperature for 30 minutes. The fusion protein DD1 will precipitate in this low pH environment. Then centrifuged at 4°C and 39,000 g for 10 minutes, and the collected precipitate was resuspended in 10 mL of ultrapure water to obtain a fusion protein DD1 suspension.

[0042] The fusion protein DD1 after induction expression and purification was analyzed by Tricine-SDS-PAGE small molecule protein gel. The results are as follows Figure 3 As shown, after IPTG induction, the band of the target fusion protein (i.e., fusion protein DD1) at around 14.4 kDa became darker, and the band of the purified fusion protein DD1 was also at this location with a higher concentration. The yield of fusion protein DD1 was determined to be about 3.5 g / L.

[0043] (IV) Enzymatic hydrolysis and purification of G-DiPGLa-H 400 U of TEV protease was added to the fusion protein DD1 suspension, and then the suspension was incubated at 30°C for 50 min, with sampling every 10 min. The samples were analyzed by Tricine-SDS-PAGE small molecule protein gel analysis. Figure 3 As shown in Figure 2, as the digestion time increases, the concentration of the fusion protein DD1 decreases due to cleavage, and DAMP4-GPGS-ENLYFQ and G-DiPGLa-H (the latter has a lower molecular weight and is not shown in the figure) are released. Figure 3 ), the fusion protein DD1 was completely cleaved at 50 min.

[0044] After 50 minutes of enzymatic hydrolysis, 10 mL of the sample was adjusted to pH 6.8 with NaOH and allowed to stand at room temperature for 30 minutes. The sample was centrifuged at 4°C and 38,000 g for 20 minutes. The supernatant and precipitate were sampled and marked, and the samples were detected by Tricine-SDS-PAGE electrophoresis. Figure 4 As shown, the 11.6 kDa DAMP4-GPGS-ENLYFQ protein band was mainly detected in the precipitate, and the 2.6 kDa protein band detected in the supernatant was the target recombinant protein G-DiPGLa-H.

[0045] The supernatant was analyzed by RP-HPLC and LC-MS. Figure 5 As shown in the figure, the HPLC results showed that the main peak of G-DiPGLa-H appeared at 8.4 minutes, and the protein purity was about 96%; the MS results revealed the consistency between the theoretical protein molecular weight of G-DiPGLa-H (2607.11 Da) and the actual molecular weight (2620.40 Da). Finally, it was concluded that 31.1 mg of G-DiPGLa-H could be purified from 1L of fermentation broth, and the yield reached 31.1 mg / L.

[0046] (V) Antibacterial and bactericidal activities of G-DiPGLa-H Take out Escherichia coli (K88, ATCC25922), Pseudomonas aeruginosa (ATCC27853), Acinetobacter baumannii (ATCC19606), and Staphylococcus aureus (ATCC29213, Mu50) from the -80℃ freezer, thaw them on ice, and streak them for overnight culture. Inoculate a single clone in 3mL LB or TSB medium and place it in a 37℃ constant temperature shaker for overnight culture (12~16h). The overnight bacterial solution is calibrated with MHB medium to a concentration of 0.5 McFarland turbidimetric standard (1~2×10 8 CFU / mL), and then dilute the corrected bacterial solution with MHB medium at 1:100. Take a sterile 96-well plate, dilute the drug (DiPGLa-H, G-DiPGLa-H or fusion protein DD1) gradiently, and add 100μL to wells 1 to 8 in sequence, and add 100μL of bacterial solution to wells 1 to 9 respectively. Set the wells with only MHB medium as blank controls. Incubate in a 37℃ incubator for 24h, observe the turbidity of the bacterial solution in each well, and measure the OD600 value with an enzyme marker to determine the minimum inhibitory concentration (MIC). Select 4μL of each well with a concentration greater than the MIC value, spot plate on solid MH medium, place in a 37℃ incubator overnight, count the number of viable bacteria, and use the killing concentration of 99.9%~99.99% in these wells as the minimum bactericidal concentration (MBC) of the drug compared with the blank control group. Repeat three rows for each bacterium, and repeat the experiment 3 times.

[0047] The minimum inhibitory concentration was used to compare the antibacterial activities of DiPGLa-H and G-DiPGLa-H. Figure 6 As shown in the figure, the minimum inhibitory concentration of the chemically synthesized antimicrobial peptide DiPGLa-H and the purified G-DiPGLa-H against Escherichia coli K88 was 8 μg / mL, and the fusion protein DD1 did not show any antibacterial activity.

[0048] In addition, the results of the antibacterial and bactericidal activities (MIC and MBC assays) of G-DiPGLa-H against other Gram-negative and Gram-positive bacteria are shown in Table 3.

[0049] Table 3. MIC (MBC) of G-DiPGLa-H and DiPGLa-H against bacteria: Unit (µg / mL)

[0050] The results show that G-DiPGLa-H A. baumannii ATCC19606 has the best antibacterial and bactericidal activity (MIC / MBC=2µg / mL) against Gram-positive bacteria S. aureus ATCC29213 has a good inhibitory effect (MIC=8µg / mL) on drug-resistant bacteria S. aureus The antibacterial activity of Mu50 was significantly stronger than that of common non-resistant bacteria (specifically S. aureus ATCC29213), and the antibacterial and bactericidal activities of G-DiPGLa-H against Mu50 were also significantly stronger than those of DiPGLa-H; overall, the biosynthesized G-DiPGLa-H had a broad spectrum of antibacterial and bactericidal activities against the tested Gram-negative and Gram-positive bacteria (including drug-resistant bacteria).

[0051] 6. Evaluation of hemolytic activity of G-DiPGLa-H 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 red blood cell pellet was aspirated and resuspended in PBS (pH 7.4) to obtain a 1% (v / v) red blood cell dilution. The red blood cell dilution and different concentrations (1-128 μg / mL) of G-DiPGLa-H were mixed in equal volumes in a 96-well plate and placed in a 37°C incubator 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 values ​​were measured at an OD value of 576 nm using an enzyme reader to evaluate the release of hemoglobin. This experiment was repeated three times, with three replicates in each experiment. The hemolytic activity test results of G-DiPGLa-H are shown in Figure 2. Figure 7 As shown in the figure, with the increase of G-DiPGLa-H concentration, G-DiPGLa-H showed lower hemolytic ability (5.1%, 7.2%) in the range of 64~128μg / mL. However, this concentration is much lower than the maximum values ​​of MIC and MBC of G-DiPGLa-H against different strains measured in (V). Therefore, the biosynthesized G-DiPGLa-H has good in vivo application value.

[0052] (VII) Application of G-DiPGLa-H in mouse intraperitoneal infection model Six to eight-week-old C57BL / 6 male mice were selected as experimental subjects. The experimental mice were divided into 8 groups: survival group and dissection group injected with PBS, survival group and dissection group injected with fusion protein DD1, survival group and dissection group injected with DiPGLa-H, and survival group and dissection group injected with G-DiPGLa-H. There were 16 mice in each survival group and 5 mice in each dissection group. At the beginning of the experiment, 8 groups of mice were intraperitoneally injected with a lethal dose of Staphylococcus aureus Mu50. 30 minutes later, 2 groups of mice were injected with G-DiPGLa-H at a concentration of 32 μg / mL, and then injected once every 6 hours for a total of 3 times. 2 groups of mice were injected with DiPGLa-H in the same way, 2 groups of mice were injected with fusion protein DD1 in the same way, and the other 2 groups of mice were injected with PBS as a control. All mice in the dissection group were killed on the second day after infection. After dissecting the mice, the heart, liver, spleen, and kidney were removed, homogenized, and diluted to 10 with PBS. 7 100 μL of the serial dilutions were spread on LBA plates and incubated at 37°C overnight. Survival rate and bacterial counts were analyzed using GraphPad Prism software. The experimental protocol and animal use were approved by the Animal Care and Use Committee of Northwest Agriculture and Forestry University.

[0053] The experimental results showed that all mice in the PBS and fusion protein DD1 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). The detection of bacterial load in mouse organs found that the bacterial load in each organ of the G-DiPGLa-H and DiPGLa-H dissection groups was significantly reduced after treatment. The bacterial load in the heart, spleen, liver, and kidney of the G-DiPGLa-H dissection group was reduced by 98.69%, 99.45%, 99.90%, and 99.92%, respectively, compared with the PBS dissection group ( Figure 8 B). Figure 8 B It can also be seen that the bacterial load in the liver and spleen of mice in the G-DiPGLa-H autopsy group was significantly lower than that in the DiPGLa-H autopsy group. These results prove that biosynthesized G-DiPGLa-H can effectively treat intraperitoneal infection in mice caused by drug-resistant bacteria Mu50, and can be used as a new antimicrobial peptide with good application prospects.

Claims

1. A DAMP4-DiPGLa-H fusion protein, characterized in that: In this fusion protein, DAMP4 and DiPGLa-H are fused via a tag linker sequence and a protease recognition sequence serving as a cleavage site.

2. A DAMP4-DiPGLa-H fusion protein, characterized in that: The amino acid sequence of the fusion protein is shown in SEQ.ID.NO.

2.

3. A recombinant antimicrobial peptide, characterized in that: The antimicrobial peptide is a DiPGLa-H recombinant protein with a cleaved protease recognition sequence at the amino terminal.

4. A recombinant antimicrobial peptide, characterized in that: The amino acid sequence of the antimicrobial peptide is shown in SEQ.ID.NO.

3.

5. A recombinant expression vector, characterized in that: The vector contains a gene sequence for encoding the DAMP4-DiPGLa-H fusion protein as claimed in claim 1 or 2.

6. A recombinant bacterium, characterized in that: The recombinant bacterium contains the recombinant expression vector as claimed in claim 5.

7. A method for preparing a DAMP4-DiPGLa-H fusion protein, characterized in that: The following steps are involved: Step 1: Design and synthesize the corresponding target gene sequence according to the amino acid sequence of the DAMP4-DiPGLa-H fusion protein, wherein the fusion protein is obtained by fusing DAMP4, a linker sequence, a protease recognition sequence and DiPGLa-H in sequence from the amino terminus to the carboxyl terminus; Step 2: Connecting the target gene sequence to the expression vector backbone to obtain a recombinant expression vector, and transforming the recombinant expression vector into a host bacterium to obtain a genetically engineered bacterium for expressing the DAMP4-DiPGLa-H fusion protein; Step 3: Using the genetically engineered bacteria, the DAMP4-DiPGLa-H fusion protein is induced to be expressed intracellularly, and then the expression product is separated and purified to obtain the DAMP4-DiPGLa-H fusion protein.

8. A method for preparing a recombinant antimicrobial peptide, characterized in that: The following steps are involved: The DAMP4-DiPGLa-H fusion protein is prepared by the preparation method of the DAMP4-DiPGLa-H fusion protein as described in claim 7; the DAMP4-DiPGLa-H fusion protein is cut by a protease compatible with the fused protease recognition sequence, and then the DiPGLa-H recombinant protein, i.e., the recombinant antimicrobial peptide, is obtained by isoelectric precipitation.

9. The method for preparing a recombinant antimicrobial peptide according to claim 8, characterized in that: The amino acid sequence of the fusion protein is shown in SEQ.ID.NO.2, and the fusion protein is cleaved by TEV protease.

10. Use of the recombinant antimicrobial peptide according to claim 3 or 4 in the preparation of a medicament for treating infectious diseases caused by Gram-negative bacteria, Gram-positive bacteria or drug-resistant bacteria.

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