Tandem expression method of antibacterial peptide mutant AMP-GN, recombinant expression vector and application

Abstract

The invention provides a tandem expression method of an antibacterial peptide mutant AMP-GN, a recombinant expression vector and application, and belongs to the technical field of molecular biology and genetic engineering. Two spaced amino acids, namely glycine and asparagine, are inserted in front of and behind the sequence of an antibacterial peptide monomer AMP, and the gene sequence of the antibacterial peptide monomer AMP is optimized, so that the antibacterial peptide monomer AMP can realize efficient transcription and translation in escherichia coli BL21 in a tandem polyploid form, and a novel antibacterial peptide mutant AMP-GN with antibacterial activity is successfully prepared through specific cutting of hydroxylamine. The compound shows remarkable bacteriostatic activity on escherichia coli and staphylococcus aureus. The antibacterial peptide mutant AMP-GN can effectively reduce the potential toxicity of the antibacterial peptide mutant AMP-GN to host cells, realizes high-yield expression, lays a solid scientific foundation for low-cost large-scale production of novel antibacterial drugs, and has a wide application prospect in the fields of foods and medicines.

Description

Technical Field

[0001] The present invention belongs to the technical fields of molecular biology and genetic engineering, and particularly relates to a tandem expression method of an antimicrobial peptide mutant AMP-GN, a recombinant expression vector and applications thereof. Background Art

[0002] Antimicrobial peptides are a class of small peptides widely present in organisms (usually composed of 12 - 50 amino acids), with broad-spectrum antibacterial activity and being an important part of the host innate immune system. Since Swedish scientists Boman et al. first isolated the antimicrobial peptide Cecropin from the silkworm pupa in 1980, thousands of antimicrobial peptides have been identified in insects, amphibians, mammals, plants and microorganisms. Their mechanism of action is significantly different from that of traditional antibiotics. These peptide molecules achieve bacteriostatic effects by disrupting the microbial cell membrane, interfering with intracellular metabolism or regulating the host immune response, etc., and thus show unique inhibitory potential against multi-drug resistant bacteria (such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), etc.).

[0003] The preparation strategies of antimicrobial peptides mainly include extraction from natural resources, chemical synthesis and biosynthesis through genetic engineering. Among them, the extraction method has low yield and high cost, and the chemical synthesis method has high purity and precise sequence control, but it will produce toxic by-products, and the synthesis of large molecular polypeptides is relatively complex. The biological method through the recombinant expression system has the advantages of low cost, high efficiency, strong applicability and environmental sustainability, and is a promising method. Escherichia coli and Bacillus subtilis have advantages such as good genetic background characteristics, fast growth rate and suitability for high-density fermentation in the biosynthesis of AMPs, and are good chassis cells for producing antimicrobial peptides. Currently, strategies such as codon optimization, fusion tag strategy and tandem expression can be used to improve the heterologous expression efficiency of antimicrobial peptides. However, problems such as easy degradation, low expression level and toxicity to host cells exist during the heterologous expression process, which limit its further application.

[0004] The present invention improves the expression level and expression efficiency of antimicrobial peptides, and at the same time improves their stability and solubility through strategies such as optimizing the gene sequence of antimicrobial peptides, tandem recombinant expression and fusion protein chaperone method. The present invention takes AMP as the research object, and the gene sequence of the antimicrobial peptide AMP is tandemly expressed in Escherichia coli BL21(DE3) through the spacer amino acids Asn-Gly and then cleaved by hydroxylamine to obtain the mutant AMP-GN of the antimicrobial peptide AMP. By increasing the gene copy number, the expression of the target polypeptide is improved, the degradation of monomeric antimicrobial peptides by proteases in host cells and the toxic effect on host cells are reduced, and bioactive monomeric peptides are released by hydroxylamine cleavage to achieve the improvement of the expression level of antimicrobial peptides, providing a theoretical basis for the industrial production of the antimicrobial peptide AMP-GN as a new antimicrobial agent. Summary of the Invention

[0005] To solve the above problems, the purpose of the present invention is to design a recombinant protein containing 2-10 tandem repeats of AMP-GN, realize the low-cost preparation of the antimicrobial peptide mutant AMP-GN in Escherichia coli, and improve the expression level by optimizing the vector and fermentation conditions. The optimized best conditions are the fusion protein composed of 10 tandem repeats, the vector used is pET21a, the best expression condition is to induce with 0.75 g / L of lactose at 31 °C for 14 h, obtain the antimicrobial peptide mutant AMP-GN by hydroxylamine cleavage, and measure the inhibitory effects of this antimicrobial peptide on Escherichia coli and Staphylococcus aureus by the micro-dilution method. This strategy effectively reduces the potential toxicity of the antimicrobial peptide mutant AMP-GN to host cells, and provides a theoretical basis for the large-scale preparation, development and utilization of this antimicrobial peptide.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a tandem expression method for the antimicrobial peptide mutant AMP-GN, comprising the following steps: tandemly connecting 2-10 gene repeat sequences of the antimicrobial peptide monomer AMP through spacer amino acids, and ligating with an expression vector to construct a recombinant vector to realize the heterologous expression of the fusion protein (AMP-GN) 2-10 -His6 in Escherichia coli, and obtaining the antimicrobial peptide mutant AMP-GN by chemical decomposition with hydroxylamine.

[0008] Preferably, the amino acid sequence of the antimicrobial peptide mutant AMP-GN is as shown in SEQ ID NO.1.

[0009] Preferably, the amino acid sequence of the antimicrobial peptide monomer AMP is as shown in SEQ ID NO.2; the spacer amino acids are glycine and asparagine.

[0010] Preferably, the heterologous expression is: the expression vector is pET21a, the final concentration of lactose is 0.75 mmol / L, the induction time is 14 hours, and the temperature is 31 °C.

[0011] Preferably, the method of chemical decomposition with hydroxylamine is: chemically cleaving the purified fusion protein with 3M hydroxylamine at 45 °C for 8 h.

[0012] The present invention also provides a recombinant expression vector. The construction method of the recombinant vector is: using homologous recombination and PCR cloning techniques, ligating the gene repeat sequences in the above-mentioned tandem expression method with the expression vectors pQE80L, pET21a and pET22b respectively, and then transforming them into Escherichia coli BL21. Through gene sequencing verification, the positive transformants screened out are the successfully constructed recombinant expression vectors.

[0013] The present invention also provides the application of the antimicrobial peptide mutant AMP-GN in the tandem expression method described in the above technical solution in food or medicine.

[0014] Preferably, the antimicrobial peptide mutant AMP-GN has significant inhibitory activity against Escherichia coli and Staphylococcus aureus and is applied in the food or medicine field.

[0015] Advantages of the present invention:

[0016] The present invention provides a tandem expression method of an antimicrobial peptide mutant AMP-GN, a recombinant expression vector and an application thereof. This method not only successfully obtains the target short peptide with bacteriostatic activity, but also effectively reduces the toxic effect of the antimicrobial peptide mutant AMP-GN on host cells. At the same time, high-yield expression of the antimicrobial peptide mutant AMP-GN is achieved, laying a solid theoretical foundation and experimental basis for the low-cost and large-scale preparation of this antimicrobial peptide. Brief Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0018] Figure 1 It is the gel electrophoresis diagram of linearized plasmids pQE80L, pET21a, pET22b and the PCR amplification products of the target gene; M: Marker; 1-3: Linearized plasmids pET22b, pET21a and pQE80L; 5-7: PCR amplification products of the target gene AMP-GN with homologous arms of pET22b, pET21a and pQE80L respectively 10 respectively;

[0019] Figure 2 It is the SDS-PAGE analysis of the preliminary expression and purification of the fusion protein; Lanes 1-2: Cell lysis solutions of the control group and the experimental group; M: Marker; 4: Effluent during the purification of the supernatant of the experimental group after cell lysis; 5: Eluate of WashBuffer during the purification of the supernatant of the experimental group after cell lysis; 6: Target protein after purification of the supernatant of the experimental group after cell lysis; 7: Cell lysis precipitate of the experimental group;

[0020] Figure 3 It is the influence of different vectors on the expression level of the fusion protein. Lanes 1-2 are the experimental group and the control group of pet28a-AMP-GN 10 protein expression respectively; 3-4 are the experimental group and the control group of pQE80L-AMP-GN 10 protein expression respectively; 5-6 are the experimental group and the control group of pET21a-AMP-GN 10Experimental and control groups for protein expression; 7-8 are pET22b-AMP-GN 10 Experimental and control groups for protein expression; M: Marker

[0021] Figure 4 Effect of lactose concentration on the expression level of the fusion protein, where 2-6 are concentrations of 0.25 g / L, 0.5 g / L, 0.75 g / L, 1 g / L, and 1.25 g / L respectively; M: Marker

[0022] Figure 5 Effect of induction time on the expression level of the fusion protein, where 2-6 are induction times of 8 h, 10 h, 12 h, 14 h, and 16 h respectively; M: Marker

[0023] Figure 6 Effect of induction temperature on the expression of the fusion protein, where 2-6 are induction temperatures of 16 °C, 21 °C, 26 °C, 31 °C, and 36 °C respectively; M: Marker

[0024] Figure 7 Liquid-phase detection chart for identifying monomeric peptides after hydroxylamine cleavage Detailed implementation methods

[0025] The present invention will be further described below in combination with specific examples and method examples. The scope of the present invention is not limited thereto. Various changes or modifications to the material components and dosages in these implementation schemes also fall within the protection scope of the present invention within the premise of the scope

[0026] The present invention provides a method for tandem expression of an antibacterial peptide mutant AMP-GN, comprising the following steps: tandemly connecting the gene repeat sequences of 2-10 antibacterial peptide monomers AMP through spacer amino acids, and ligating with an expression vector to construct a recombinant vector to achieve the heterologous expression of the fusion protein (AMP-GN) 2-10 -His6 in Escherichia coli, and obtaining the antibacterial peptide mutant AMP-GN through chemical decomposition with hydroxylamine

[0027] Further, the amino acid sequence of the antibacterial peptide mutant AMP-GN is as shown in SEQ ID NO.1

[0028] Further, the amino acid sequence of the antibacterial peptide monomer AMP is as shown in SEQ ID NO.2; the spacer amino acids are glycine and asparagine

[0029] Further, the heterologous expression is: the expression vector is pET21a, the final concentration of lactose is 0.75 mmol / L, the induction time is 14 hours, and the temperature is 31 °C

[0030] Further, the method for chemical decomposition of hydroxylamine is as follows: The purified fusion protein is chemically cleaved with 3M hydroxylamine at 45°C for 8 hours.

[0031] The present invention also provides a recombinant expression vector. The construction method of the recombinant vector is as follows: Using homologous recombination and PCR cloning techniques, the gene repeat sequences in the tandem expression method described in the above technical solution are respectively ligated with the expression vectors pQE80L, pET21a, and pET22b, and then transformed into Escherichia coli BL21. Through gene sequencing verification, the positive transformants screened out are the successfully constructed recombinant expression vectors.

[0032] The present invention also provides the application of the antimicrobial peptide mutant AMP-GN in the tandem expression method described in the above technical solution in food or medicine.

[0033] Further, the antimicrobial peptide mutant AMP-GN has significant inhibitory activity against Escherichia coli and Staphylococcus aureus and is applied in the fields of food or medicine.

[0034] The present invention provides a preparation method of an antimicrobial peptide AMP-GN.

[0035] Furthermore, the preparation method of the antimicrobial peptide mutant AMP-GN is achieved through the following steps:

[0036] 1) Repeat and concatenate the antimicrobial peptide mutant AMP-GN sequence, add a His6 tag and a stop codon (TAA) to the N-terminus of the concatenated sequence, and then perform prokaryotic expression;

[0037] 2) Design seamless cloning primers to perform PCR amplification to obtain gene fragments for cloning and ligation;

[0038] 3) Design seamless cloning primers to linearize the plasmids pQE80L, pET21a, and pET22b by reverse PCR, and recover the linearized plasmids;

[0039] 4) Through the seamless cloning system, react the linearized plasmid and the gene obtained by PCR amplification to obtain the recombinant plasmids pQE80L-AMP-GN 10 、pET21a-AMP-GN 10 、pET22b-AMP-GN 10 ;

[0040] 5) Perform prokaryotic induction expression on the tandem fusion protein of the antimicrobial peptide mutant AMP-GN:

[0041] The recombinant plasmids pQE80L-AMP-GN 10 、pET21a-AMP-GN 10, pET22b-AMP-GN 10 Transform it into competent Escherichia coli cells BL21, and respectively pick positive bacteria on the LB solid medium containing resistance;

[0042] Perform prokaryotic induction expression on the positive bacteria: the expression vectors are pET28a, pQE80L, pET21a, pET22b, the temperature is 16 - 36 °C, the lactose concentration is 0.25 - 1.25 g / L, and the induction time is 8 - 16 h.

[0043] Furthermore, the expression vectors are respectively pET28a, pQE80L, pET21a, pET22b; the induction temperatures are 16 °C, 21 °C, 26 °C, 31 °C, 36 °C; the induction times are 8 h, 10 h, 12 h, 14 h, 16 h; the lactose concentrations are 0.25 g / L, 0.5 g / L, 0.75 g / L, 1 g / L, 1.25 g / L.

[0044] Furthermore, after expressing the protein under the optimal conditions of the present invention, lyse the cells, purify the fusion protein with His tag using a nickel column, cleave the fusion protein with hydroxylamine at a volume concentration of 3 M, dialyze to remove hydroxylamine to obtain the cleavage product, and detect the antibacterial peptide AMP-GN monomer obtained by cleavage through liquid phase;

[0045] Furthermore, perform antibacterial activity determination on the antibacterial peptide AMP-GN monomer obtained according to the present invention. Dissolve the antibacterial peptide AMP-GN after removing hydroxylamine with 20 mmol / L PBS, use 20 mmol / L PBS as a negative control, and detect the antibacterial activity of the antibacterial peptide AMP-GN.

[0046] According to the antibacterial effect of the antibacterial peptide mutant AMP-GN obtained in the present invention, it can be used as a preservative to inhibit pathogenic bacteria in food and extend the shelf life of meat, dairy products, and ready-to-eat foods. Replace chemical preservatives (such as potassium sorbate) to meet consumers' demands for natural and safe foods; it can also develop antibacterial peptide dressings or gels to accelerate the healing of wounds such as burns and chronic ulcers and inhibit bacterial growth.

[0047] In the present invention, the amino acid sequence of the antibacterial peptide mutant AMP-GN is as shown in SEQ ID NO.1: GVDKPPYLPRPRPIRRPGGRN.

[0048] In the present invention, the amino acid sequence of the antibacterial peptide monomer AMP is as shown in SEQ ID NO.2: VDKPPYLPRPRPIRRPGGR.

[0049] Example 1

[0050] Preparation of recombinant strain

[0051] 1) Preparation of fusion protein: The antimicrobial peptide mutant AMP-GN with the sequence GVDKPPYLPRPRPIRRPGGRN (SEQ ID No.1) was repeatedly ligated 10 times to obtain a tandem expression cassette. A His6 tag and a stop codon (TAA) were added to the tandem sequence to obtain the fusion protein (AMP-GN) 10 -His6 nucleotide sequence was submitted to the company for synthesis on the vector pET28a

[0052] 2) Preparation of target gene fragment: The seamless cloning primers were designed as follows (the bold part is the gene fragment homologous to the ligation site of the target vector):

[0053] Table 1 Gene sequences

[0054]

[0055] Using the vector pET-28a containing the target gene as a template, the fusion protein (AMP-GN) 10 gene fragment was amplified by PCR with specific primers, and the target gene fragment was obtained by gel cutting and recovery

[0056] The PCR reaction system and amplification conditions are as follows:

[0057] Table 2 PCR reaction system and amplification conditions

[0058] Component Volume (μL) Forward primer F 2.5 μL Reverse primer R 2.5 μL <![CDATA[pET28a-AMP-GN 10 > 2.5 μL GoldStarMax 25 μL <![CDATA[ddH2O]]> 17.5 μL

[0059] The amplification program was: pre-denaturation at 95°C for 10 min; denaturation at 94°C for 30 s, annealing at 53°C for 30 s, extension at 72°C for 24 s, 30 cycles of reaction; extension at 72°C for 10 min

[0060] 3) Preparation of target vector: Using the plasmids containing pQE80L, pET21a, and pET22b vectors in the laboratory as templates, the pQE80L, pET21a, and pET22b vectors were linearized by PCR amplification with specific primers, and the linearized plasmids pQE80L, pET21a, and pET22b were obtained by gel cutting and recovery

[0061] The seamless cloning primers were designed as follows:

[0062] Table 3 Seamless cloning primers

[0063]

[0064]

[0065] 4) Use homologous recombinase to ligate the PCR product of the target sequence with the linearized plasmids pQE80L, pET21a, and pET22b in a 50°C water bath for 20 min. Transform the ligation product into Escherichia coli BL21 competent cells, spread them on an LB solid plate containing kanamycin sulfate resistance, and incubate in a 37°C incubator for 12 h. Transfer the transformants grown on the plate to a test tube and extract the plasmid. Send the plasmid to Genewiz Suzhou Co., Ltd. for sequencing. The sequencing results show success, and the recombinant strains are BL21(pQE80L-AMP-GN 10 ), BL21(pET21a-AMP-GN 10 ), BL21(pET22b-AMP-GN 10 ).

[0066] Example 2

[0067] Expression and purification of the fusion protein (AMP-GN) 10

[0068] Activate the recombinant strain (pET28a-AMP-GN 10 ), incubate at 37°C for 12 h, pick a single colony and inoculate it into 5 mL of LB liquid medium containing kanamycin sulfate (100 mg / mL) for scale-up culture. Inoculate the bacterial solution into 50 mL of LB liquid medium with kanamycin sulfate, and incubate with shaking at 37°C until OD 600 = 0.6 - 0.8. Add lactose to a final concentration of 1 g / L and induce overnight at 37°C. At the same time, set up a bacterial solution without added lactose as a control group. Centrifuge at 8000 rpm for 5 min at room temperature to collect the bacteria. Resuspend the bacteria with 5 mL of Lysis Buffer, sonicate, and then centrifuge to obtain the disrupted precipitate and supernatant. Filter the supernatant through a sterile filter membrane and bind it to Ni2+ resin in an ice-water bath for 60 - 90 min. Pour the protein solution bound to Ni 2+ resin into a purification chromatography column, and wash the miscellaneous proteins on the resin with 10 mL of Wash Buffer (100 mM imidazole, 20 mM Tris-HCl, 1 mM dithiothreitol, 200 mM NaCl); finally, elute the target protein with His tag with 5 mL of Elution Buffer (500 mM imidazole, 20 mM Tris-HCl, 1 mM dithiothreitol, 200 mM NaCl). Take samples respectively, resuspend the disrupted precipitate with sterile water, take 20 uL of each sample and add 5 ul of 5× loading buffer, boil at 100°C for 10 min, and collect the supernatant for 12% SDS-PAGE electrophoresis detection. There is a band at the size of the target protein in the experimental group, and none in the control group. According to the electrophoresis detection results, it can be known that the fusion protein is successfully expressed.

[0069] Example 3​

[0070] Hydroxylamine cleavage of the fusion protein

[0071] Collect the purified fusion protein, cleave it with hydroxylamine solution at a final concentration of 3 M at 45 °C for 8 h, dialyze to remove hydroxylamine from the solution, and lyophilize the solution to obtain the antimicrobial peptide mutant AMP-GN.

[0072] Comparative Example 1

[0073] Fusion protein (AMP-GN) 10 Screening of the expression vector.

[0074] The constructed recombinant plasmids pQE80L-AMP-GN 10 、pET21a-AMP-GN 10 、pET22b-AMP-GN 10 were transformed into the expression host competent cell E. coli BL21(DE3), expressed according to the method in Example 2, detected by 12% SDS-PAGE electrophoresis, and the best expression conditions were judged according to the intensity of the bands. The results showed that pET21a as the expression vector had the best result.

[0075] Comparative Example 2

[0076] Fusion protein (AMP-GN) 10 Screening of the expression conditions.

[0077] The recombinant plasmid pET21a-AMP-GN 10 was expanded in culture. To determine the best expression conditions of the fusion protein, a series of different induction conditions were set, including lactose concentration (0.25 g / L, 0.5 g / L, 0.75 g / L, 1 g / L, 1.25 g / L), induction time (8 h, 10 h, 12 h, 14 h, 16 h), and induction temperature (16 °C, 21 °C, 26 °C, 31 °C, 36 °C). It was expressed according to the method in Example 2, and the best conditions were judged according to the SDS-PAGE electrophoresis pattern. The results showed that the protein expression was the highest under the conditions of lactose concentration of 0.75 g / L, induction time of 14 h, and induction temperature of 31 °C.

[0078] Method Example 1

[0079] Liquid phase detection of the antimicrobial peptide mutant AMP-GN

[0080] The antibacterial peptide mutant AMP-GN after cleavage was detected by high performance liquid chromatography (HPLC). The desalted and freeze-dried sample was dissolved in 20 mmol / L PBS buffer, and the sample was filtered for impurities using a 0.22 μm aqueous filter membrane to prevent column blockage. The chromatographic detection conditions were as follows: mobile phase A was ultrapure water containing 0.1% TCA, and mobile phase B was acetonitrile containing 0.1% TCA; column temperature: 25 °C; the mobile phase was degassed by ultrasonic treatment for 30 min. The detector was an ultraviolet-visible detector; the injection volume was 20 μL; the detection wavelength was 214 nm. The chromatographic column was first equilibrated with mobile phase B, and then a gradient elution program was set. The gradient elution conditions are shown in Table 4 below.

[0081] Table 4 Gradient Elution Conditions

[0082] Time (min) Aqueous phase percentage (%) Flow rate (mL / min) 0.0 85 1 0.01 85 1 15.0 70 1 20 85 1

[0083] Method Example 2

[0084] Antibacterial Activity Assay

[0085] The antibacterial peptides (antibacterial peptide mutant AMP-GN) after liquid phase detection were serially diluted: In a sterile 1.5 mL ep tube, 100 μL of the antibacterial peptide stock solution was pipetted into a test tube containing 100 μL of medium using a sterile pipette and mixed well. 100 μL of the solution was pipetted from the 200 μL test tube into the next test tube containing 100 μL of medium and mixed. And so on for serial dilution. Escherichia coli ATCC25922 and Staphylococcus aureus ATCC25923 were inoculated into 5 mL of MH medium in advance and cultured for 6 h, and then diluted to 0.5 McFarland turbidity (OD600 = 0.08 - 0.13) with MH medium in a laminar flow hood respectively. 100 μL of the diluted bacterial solution was added to each well of a 96-well plate.

[0086] Addition of antibacterial peptide solution: The above-mentioned diluted antibacterial peptide solutions with different concentrations were successively added to the corresponding wells of the 96-well plate, 100 μL per well, so that the final total volume in each well was 200 μL, and mixed well by pipetting. At the same time, positive control wells (containing only bacterial solution and medium, without antibacterial peptide) and negative control wells (containing only medium, without bacterial solution and antibacterial peptide) were set, and 3 replicate wells were set for each concentration. After sealing the 96-well plate with a sealing film, it was placed in an incubator at 37 °C for 16 - 24 hours. Observe the turbidity of each well to judge the bacterial growth. The negative control wells should be clear and transparent, and the positive control wells should be turbid. In the wells containing antibacterial peptide, if the solution is clear and transparent, it indicates that bacterial growth is inhibited, and the concentration of the antibacterial peptide at which the first clear well appears is the MIC (Table 5).

[0087] Table 5 Results

[0088]

[0089] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tandem expression method for an antibacterial peptide mutant AMP-GN, characterized in that, Comprising the following steps: The gene repeat sequences of 2-10 antimicrobial peptide monomers AMP are tandemly connected by spacer amino acids and ligated with an expression vector to construct a recombinant vector, thereby achieving the heterologous expression of the fusion protein (AMP-GN) 2-10 -His6 in Escherichia coli, and an antimicrobial peptide mutant AMP-GN is obtained through chemical decomposition with hydroxylamine 2. The tandem expression method according to claim 1, characterized in that, The amino acid sequence of the antimicrobial peptide mutant AMP-GN is shown in SEQ ID NO.

1.

3. The tandem expression method according to claim 1, wherein The amino acid sequence of the antimicrobial peptide monomer AMP is shown in SEQ ID NO.2; the spacer amino acids are glycine and asparagine.

4. The tandem expression method according to claim 1, wherein The heterologous expression is as follows: the expression vector is pET21a, the final concentration of lactose is 0.75 mmol / L, the induction time is 14 hours, and the temperature is 31 °C.

5. The tandem expression method according to claim 1, wherein The method for chemical decomposition by hydroxylamine is as follows: the purified fusion protein is chemically cleaved with 3M hydroxylamine at 45 °C for 8 h.

6. A recombinant expression vector, characterized in that, The method for constructing the recombinant vector is as follows: using homologous recombination and PCR cloning techniques, the gene repeat sequences in the tandem expression method described in claim 1 are respectively ligated to the expression vectors pQE80L, pET21a, and pET22b, and then transformed into Escherichia coli BL21. Through gene sequencing verification, the positive transformants screened out are the successfully constructed recombinant expression vectors.

7. Use of the antimicrobial peptide mutant AMP-GN in the tandem expression method described in claim 1 in food or medicine.

8. The application according to claim 7, wherein The antimicrobial peptide mutant AMP-GN has significant inhibitory activity against Escherichia coli and Staphylococcus aureus and is applied in the food or medicine field.

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