Polypeptide for promoting exocrine expression of target protein, biological material and application thereof, antibacterial peptide fusion protein and recombinant genetic engineering bacterium

Abstract

The invention belongs to the technical field of biological genetic engineering, and particularly relates to a polypeptide for promoting exocrine expression of a target protein, a biological material and application thereof, an antibacterial peptide fusion protein and a recombinant genetic engineering bacterium. As a signal peptide, the polypeptide provided by the invention further improves the exocrine expression efficiency and yield of the target protein; a contrast experiment verifies that compared with the existing signal peptide, the optimized and modified signal peptide has the advantage that the efficiency of exocrine expression of target protein of a constructed lactococcus lactis expression system is improved by 2.1-13.6 times. The recombinant genetically engineered bacterium constructed by the invention successfully realizes efficient exocrine expression of the antibacterial peptide by the lactococcus lactis, has the probiotic property of lactic acid bacteria and the antibacterial effect of the antibacterial peptide, can be directly orally taken or used as an additive to be applied to food and animal feed, and greatly saves the production and preparation cost of the antibacterial peptide.

Description

Technical Field

[0001] The invention belongs to the technical field of biological genetic engineering, and specifically relates to polypeptides for promoting the exocrine expression of target proteins, biological materials and applications thereof, antimicrobial peptide fusion proteins, and recombinant genetic engineering bacteria. Background Art

[0002] Antibiotics play an important role in preventing animal diseases, regulating immunity, and promoting growth. However, bacterial resistance caused by the abuse and misuse of antibiotics has seriously threatened human life and health, and the demand for the development of different antibiotic substitutes is increasing. Antimicrobial peptides (AMPs) are a class of small molecule short peptides that are widely present in organisms. They are an important part of the natural immune system. They have the characteristics of broad spectrum, high efficiency, low toxicity, and low resistance to bacteria. Therefore, they have become one of the best substitutes for antibiotics recognized internationally. Proline rich antimicrobial peptides (Pr AMPs) have attracted much attention from researchers due to their simple structure, strong antimicrobial activity against Gram-negative bacteria, and low toxicity to eukaryotic cells.

[0003] At present, the methods for obtaining antimicrobial peptides are mainly through direct separation and purification from organisms, in vitro chemical synthesis, and induction of antimicrobial peptide expression by genetically engineered bacteria. Among them, direct separation and purification from organisms is the main way to discover natural antimicrobial peptides. However, the cumbersome separation process, low separation volume, and high purification cost limit this method to be unsuitable for large-scale production; in vitro chemical synthesis is the main source of antimicrobial peptides for laboratory research and development. However, due to high synthesis costs and technical limitations, some complex antimicrobial peptides cannot currently be synthesized in vitro. This method is also not suitable for large-scale production of antimicrobial peptides. Inducing the expression of antimicrobial peptides by genetically engineered bacteria is an important method for obtaining antimicrobial peptides in large quantities. By constructing a suitable expression system, antimicrobial peptides can be produced on a large scale for clinical disease prevention and control.

[0004] Lactic acid bacteria are a type of Gram-positive bacteria that do not produce endotoxins, spores, are peroxidase-negative, and metabolize carbohydrates to produce lactic acid. Currently, only one protease (HtrA) has been found outside the lactic acid bacteria cell, which makes the expression of exogenous proteins relatively stable. Lactic acid bacteria are non-toxic and have probiotic effects in the body, and are recognized as safe microorganisms (Generally Recognized as Safe, GRAS) by the US Food and Drug Administration (FDA). Therefore, using lactic acid bacteria to secrete Pr AMPs, the recombinant bacteria have both the probiotic properties of lactic acid bacteria and the killing effect of PrAMPs on Gram-negative bacteria, which is an ideal way to use antimicrobial peptides clinically. The Nisin-Controlled gene Expression system (NICE) system is a commonly used exogenous protein expression system for lactic acid bacteria. The NICE system consists of three parts: lactic acid bacteria containing nisR and nisK genes, Nisin or its analogs as inducers, and expression vectors containing nisA or nisF promoters. Nisin is a small peptide produced by some lactococci. It can induce the transcription of nisin biosynthesis gene cluster under the control of nisA or nisF promoter through a typical two-component regulatory system. The two-component regulatory system consists of histidine kinase NisK and effector protein NisR encoded by nisK and nisR genes. NisK recognizes sub-inhibitory amounts of Nisin and then phosphorylates NisR. NisR, as an effector regulatory protein, activates the transcription of nisA or nisF promoter, allowing the target gene to be expressed. However, the low efficiency of antimicrobial peptide secretion in lactic acid bacteria expression system is still a key factor restricting the development of the industry. How to solve the inhibitory growth performance of antimicrobial peptides on lactic acid bacteria. How to further improve the secretion expression performance of lactic acid bacteria as an expression vector is a core technical problem that needs to be solved urgently. Existing technical research has found that the Usp45 signal peptide sequence can successfully secrete and express a variety of exogenous proteins. By optimizing and improving the artificial peptide sequence of the wild-type Usp45 signal peptide, the efficiency of lactic acid bacteria in secreting and expressing exogenous proteins can be further improved. Summary of the invention

[0005] In order to overcome the defects of the prior art, one of the purposes of the present invention is to provide a polypeptide for promoting the exocrine expression of a target protein as a functional element of an exogenous protein expression system of lactic acid bacteria, thereby further improving the exocrine expression efficiency.

[0006] The second purpose of the present invention is to provide a biomaterial that can achieve efficient exocrine expression of exogenous proteins, especially antimicrobial peptides.

[0007] The third object of the present invention is to provide applications of the biomaterial of the present invention.

[0008] The fourth object of the present invention is to provide an antimicrobial peptide fusion protein that can be efficiently expressed in lactic acid bacteria expression system and exhibit antimicrobial activity after the action of enterokinase in vitro.

[0009] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0010] A polypeptide for promoting the exocrine expression of a target protein, wherein the amino acid sequence thereof comprises the sequence shown in SEQ ID NO.1.

[0011] Preferably, the carboxyl terminus of the polypeptide is linked to a secretion enhancing peptide.

[0012] In a specific embodiment of the present invention, the amino acid sequence of the polypeptide is the sequence shown in SEQ ID NO.2.

[0013] A biomaterial, wherein the biomaterial is a biomaterial related to the polypeptide, and is any one of the following 1) to 4):

[0014] 1) A nucleic acid molecule encoding the above polypeptide;

[0015] 2) an expression cassette containing the nucleic acid molecule described in 1);

[0016] 3) a recombinant vector containing the expression cassette described in 2);

[0017] 4) A recombinant protein expression system comprising 1) the nucleic acid molecule, 2) the expression cassette, or 3) the recombinant vector.

[0018] In a specific embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.3.

[0019] Preferably, the recombinant protein expression system is a prokaryotic microbial expression system.

[0020] Further preferably, the recombinant protein expression system is a Lactococcus lactis expression system. In a specific embodiment of the present invention, the Lactococcus lactis is NZ9000.

[0021] The above polypeptide or biological material can be used to prepare the target protein by exocrine expression, which can improve the secretion expression efficiency and the yield of the target protein. Specifically, the above polypeptide or biological material can be used to construct a recombinant Lactococcus lactis that exocrinely expresses the target protein, for example, to construct a Lactococcus lactis that exocrinely expresses an antimicrobial peptide.

[0022] Preferably, the constructed Lactococcus lactis exocrinely expresses USP 45 The fusion protein of protein and antimicrobial peptide is preferably connected to USP by a peptide chain that can be recognized by enterokinase.45 The C-terminus of the protein.

[0023] Beneficial effects of the present invention:

[0024] 1) The present invention improves the existing USP 45 The amino acid sequence of the signal peptide is optimized and modified, and used as a signal peptide for the exocrine expression of the target protein in the prokaryotic microorganism, especially the Lactococcus lactis expression system, to further improve the exocrine expression efficiency and yield of the target protein; in the specific embodiment of the present invention, a comparative experiment is designed to verify that the USP optimized and modified by the present invention 45 Compared with the existing signal peptides, the efficiency of the exocrine expression of the target protein by the constructed Lactococcus lactis expression system is increased by 2.1 to 13.6 times.

[0025] 2) Further, the present invention constructs USP 45 and antimicrobial peptide fusion proteins, specifically USP 45 +BSN-25 fusion protein, antimicrobial peptide can recognize the broken peptide chain through enterokinase and connect to USP 45 The C-terminus of the protein, the fusion protein has no antimicrobial peptide activity after exocrine expression, does not produce growth inhibition effect on Lactococcus lactis, and ensures the large-scale proliferation of lactic acid bacteria during fermentation. After being acted on by enterokinase, the fusion protein releases antimicrobial peptides to exert the original activity of antimicrobial peptides. The present invention successfully achieves efficient exocrine expression of antimicrobial peptides by Lactococcus lactis, and the constructed recombinant Lactococcus lactis has both the probiotic properties of lactic acid bacteria and the antimicrobial effect of antimicrobial peptide BSN-25, which can be directly taken orally or used as an additive in food and animal feed, greatly saving the production and preparation cost of antimicrobial peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 PCR identification for NZ9000 pNZ8148:usp45+bsn-25;

[0027] Figure 2 The recombinant plasmid pNZ8148-M:usp45+bsn-25 was double-digested;

[0028] Figure 3 Western blot comparison of different genetically engineered bacteria expressing fusion protein USP 45 +The amount of BSN-25;

[0029] Figure 4 Western blot analysis of the expression of fusion protein USP induced by different temperatures 45 +BSN-25 quantity;

[0030] Figure 5 Western blot analysis of different Nisin concentrations to induce expression of fusion protein USP45 +BSN-25 quantity;

[0031] Figure 6 Western blot analysis of the expression of fusion protein USP induced at different times 45 +BSN-25 quantity;

[0032] Figure 7 Fusion protein USP 45 + Electrophoresis of BSN-25 before and after enterokinase digestion;

[0033] Figure 8 Fusion protein USP 45 + Antibacterial activity detection of BSN-25 before and after enterokinase cleavage. DETAILED DESCRIPTION

[0034] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto;

[0035] The specific operations of the protein separation and identification methods SDS-PAGE and Western blot used in the following examples are as follows:

[0036] SDS-PAGE experiment: The overnight culture of genetically engineered bacteria was subcultured in 200 mL GM17 at a ratio of 2% and cultured at 30°C until OD 600 =0.6, add the inducer Nisin and let it stand for cultivation. Then, the bacterial solution was separated by centrifugation at 8000rpm. Among them, the bacterial precipitate was suspended with 5mL PBS (10mmol / L). The supernatant collected by centrifugation was filtered with a 0.45μm filter membrane, concentrated to about 10mL in a dialysis bag using PEG 6000, and 5 times the volume of ice acetone (concentration 50%) was added to the concentrated supernatant, 4℃, overnight. Then, centrifuged at 4℃ and 12000rpm for 30min, the collected precipitate was placed in a ventilated place for 10min to remove residual acetone. Add 100μL of pure water to suspend the precipitate. Take 10μL 5× protein loading buffer and add it to 40μL bacterial suspension and culture solution protein acetone concentrated suspension respectively, heat in a 100℃ metal bath for 10min to fully denature the protein, centrifuge at 3000rpm for 2min, and take 25μL supernatant for SDS-PAGE.

[0037] Western blot experiment: After the protein of genetically engineered bacterial culture fluid and bacterial protein were separated by SDS-PAGE as described above, the protein to be tested was transferred to polyvinylidene fluoride (PVDF) membrane by semi-dry method, blocked with PBST buffer solution containing 5% skim milk on a constant temperature shaker for 1 hour, incubated with His-Tag Monoclonalantibody primary antibody at 4°C for 10 hours, washed with PBST buffer for 30 minutes, and then added with horseradish enzyme-labeled goat anti-mouse IgG (H+L) secondary antibody, incubated on a constant temperature shaker for 1 hour, washed with PBST for 30 minutes, and exposed to identify the expression of the target protein.

[0038] Example 1 Construction of fusion protein USP 45 + recombinant plasmid of BSN-25 expression cassette

[0039] This embodiment provides a recombinant plasmid containing a fusion protein expression cassette, and the specific construction method includes:

[0040] 1) From N-terminus to C-terminus, USP 45 protein, enterokinase-recognizable cleavage peptide chain, BSN-25 antimicrobial peptide (its amino acid sequence is shown in SEQ ID NO.5, and its encoding nucleotide sequence is shown in SEQ ID NO.6), design the amino acid sequence of the fusion protein, design its encoding nucleotide sequence according to the amino acid sequence of the fusion protein and synthesize it artificially, and its nucleotide sequence is shown in SEQ ID NO.4;

[0041] Alternatively, the gene encoding the lactic acid bacteria exoprotein USP45 is amplified and recovered by PCR, and the nucleotide sequence encoding the enterokinase-recognizable cleavage peptide chain and the nucleotide sequence encoding the BSN-25 antimicrobial peptide are sequentially connected to the USP45 by overlapping extension PCR. 45 3' end of the gene;

[0042] 2) The fusion protein encoding nucleotide sequence constructed in step 1) was connected to the plasmid vector pN8148 6×His to construct a recombinant plasmid containing a fusion protein expression cassette, which was recorded as pN8148 6×His:usp+bsn-25.

[0043] Example 2 This example constructs a Lactococcus lactis that exocrinely expresses a fusion protein using the polypeptide of the present invention as a signal peptide. This example uses a Lactococcus lactis that exocrinely expresses a fusion protein, and the construction method includes:

[0044] 1) According to the optimized and modified signal peptide amino acid sequence, as shown in SEQ ID NO.2, the encoding nucleotide sequence thereof is designed, as shown in SEQ ID NO.3, and the nucleotide sequence is artificially synthesized;

[0045] 2) using a plasmid kit to extract pNZ8148 6×His:usp+bsn-25 provided in Example 1, the plasmid was double-digested with BgII and Kpn1 to remove its promoter region, and then connected with the nucleotide sequence prepared in step 1) by the same double-digestion to construct a recombinant plasmid, which was recorded as pNZ8148-M:usp45+bsn-25;

[0046] 3) The recombinant plasmid was identified by PCR and double enzyme digestion (such as Figure 1 and Figure 2 ), the PCR product was sent for sequencing, and the pNZ8148-M:usp45+bsn-25 with correct sequence was electrotransfected into lactic acid bacteria NZ9000. The transformants were screened on GM17 plates containing 10 μg / mL chloramphenicol and identified by PCR amplification and recorded as NZ9000 pNZ8148-M:usp45+bsn-25.

[0047] Example 3

[0048] According to the same method as in Example 2, recombinant plasmids containing existing signal peptide molecule expression cassettes were constructed, namely pNZ8148-L:usp45+bsn-25, whose signal peptide amino acid sequence is shown in SEQ ID NO.7, and whose signal peptide encoding nucleotide sequence is shown in SEQ ID NO.8; pNZ8148-N:usp45+bsn-25, whose signal peptide amino acid sequence is shown in SEQ ID NO.7, and whose encoding nucleotide sequence is shown in SEQ ID NO.9. According to the same method as in Example 2, corresponding lactic acid bacteria NZ9000 expressing fusion protein exocytically were constructed, respectively recorded as NZ9000 pNZ8148:usp45+bsn-25 (the starting plasmid provided in Example 1), NZ9000 pNZ8148-L:usp45+bsn-25, and NZ9000 pNZ8148-N:usp45+bsn-25.

[0049] Example 4 Performance Characterization of the Lactobacillus Lactococcus Exocrine Expression Fusion Protein Constructed in Example 2 and Example 3

[0050] The four genetically engineered bacteria NZ9000 pNZ8148 6×His::usp+bsn-25, NZ9000 pNZ8148-L:usp45+bsn-25, NZ9000 pNZ8148-M:usp45+bsn-25 and NZ9000 pNZ8148-N:usp45+bsn-25 provided in Example 2 and Example 4 secreted and expressed the fusion protein USP under the same conditions. 45+BSN-25, the specific conditions were 80 ng / mL Nisin, static culture at 26°C for 6 h to induce the expression of the fusion protein.

[0051] Western-blot was performed using 6×His monoclonal antibody to identify USP in the culture fluid and cells of each genetically engineered bacteria. 45 +BSN-25, and compared the expression levels of their fusion proteins. The results are as follows Figure 3 As shown, Figure A shows the expression level of the fusion protein in the culture medium; Figure B shows the expression level of the fusion protein in the cell, where M, pre-stained protein Marker 10-180KDa; 1, genetically engineered bacteria NZ9000pNZ8148; 2, genetically engineered bacteria NZ9000 pNZ8148 6×His: usp45+bsn-25; 3, genetically engineered bacteria NZ9000pNZ8148-L: usp45+bsn-25; 4, genetically engineered bacteria NZ9000 pNZ8148-M: usp45+bsn-25; 5, genetically engineered bacteria NZ9000 pNZ8148-N: usp45+bsn-25. Figure 3 The results showed that the fusion protein content in the culture medium of the genetically engineered bacteria NZ9000 pNZ8148-M:usp45+bsn-25 was the highest, and compared with other genetically engineered bacteria, it could improve the exocrine expression efficiency of the fusion protein. Specifically, compared with NZ9000 pNZ8148 6×His:usp45+bsn-25, the exocrine expression efficiency of the fusion protein was increased by 32.85 times, and compared with NZ9000 pNZ8148-L:usp45+bsn-25, the exocrine expression efficiency of the fusion protein was increased by 2.13 times; compared with NZ9000 pNZ8148-N:usp45+bsn-25, the exocrine expression efficiency of the fusion protein was increased by 13.62 times.

[0052] Example 5 Optimization of conditions for exocrine expression of fusion protein of genetically engineered bacteria NZ9000 pNZ8148-M: usp45+bsn-25

[0053] Optimization method: The following optimization was performed on the induction expression conditions of lactic acid engineering bacteria NZ9000 pNZ8148-M:usp45+bsn-25 by SDS-PAGE and Western blot methods:

[0054] The overnight culture of genetically engineered bacteria NZ9000 pNZ8148-M:usp45+bsn-25 was subcultured in four 200 mL GM17 cells at a ratio of 2% and cultured at 30°C until OD 600=0.6, and add the inducer Nisin (final concentration 5 ng / mL). Then, the four culture bottles were placed in 22°C, 26°C, 30°C and 37°C incubators for 6 h. 45 +BSN-25 was used for SDS-PAGE separation and Western blot identification. The identification results are attached. Figure 4 As can be seen from the figure, the fusion protein USP secreted by the genetically engineered bacteria NZ9000 pNZ8148-M:usp45+bsn-25 in an environment of 26°C 45 +BSN-25 is relatively large in quantity.

[0055] Genetically engineered bacteria NZ9000 pNZ8148-M:usp45+bsn-25 were cultured as described above, and five 200 mL GM17 cultures were incubated at 30°C until OD 600 =0.6. Then, different amounts of inducer Nisin were added to 5 culture bottles, with final concentrations of 1ng / mL, 2.5ng / mL, 5ng / mL, 10ng / mL and 20ng / mL, respectively. All engineered bacteria were placed in a 26°C environment for 6 hours. 45 +BSN-25 was used for SDS-PAGE separation and Western blot identification. The identification results are attached. Figure 5 As can be seen from the figure, the amount of fusion protein secreted by the genetically engineered bacteria NZ9000pNZ8148-M:usp45+bsn-25 at 26°C induced by Nisin at a final concentration of 2.5 ng / mL was relatively large.

[0056] Genetically engineered bacteria NZ9000 pNZ8148-M:usp45+bsn-25 were cultured as described above. Five 200 mL GM17 culture bottles were incubated at 30°C until OD 600 =0.6. Subsequently, 2.5 ng / mL of Nisin was added to the five culture solutions and placed in an environment at 26°C for induction culture for 4 h, 6 h, 8 h, 10 h and 12 h, respectively. After the induction culture, the induced bacteria were treated according to the method described in Example 2 to treat the fusion protein USP in the bacteria and the culture solution. 45 +BSN-25 for SDS-PAGE separation and Western blot identification, the identification results are attached Figure 6 As can be seen from the figure, when the genetically engineered bacteria NZ9000pNZ8148-M:usp45+bsn-25 was induced with Nisin at a final concentration of 2.5 ng / mL for 6 h at 26°C, the amount of fusion protein secreted was relatively large.

[0057] Example 6 Fusion Protein USP 45 +BSN-25 activity detection

[0058] 1. Exocrine expression of fusion protein USP according to the conditions optimized in Example 5 45 +BSN-25, Purified USP 45 +BSN-25 was digested in vitro with enterokinase at 4°C for 24 h according to the instructions. Then, 40 μL of the digestion solution and fusion protein USP were taken respectively. 45 +BSN-25 was mixed with 10μL 5× protein loading buffer and placed in a 100℃ metal bath for 10 min to fully denature the protein. 25μL of the denatured protein solution was taken for SDS-PAGE analysis. The electrophoresis results are shown in the attached Figure 7 , where M: pre-stained protein marker 10-180KDa; 1, expressed and purified USP 45 +BSN-25; 2, USP after enterokinase digestion 45 +BSN-25. As can be seen from the figure, after the fusion protein was digested with enterokinase, part of the antimicrobial peptide BSN-25 had dissociated from the fusion protein; the fusion protein dissociated into USP45 (attached Figure 7 Lane 2) and antimicrobial peptide BSN-25 (small molecular weight protein, about 5 kDa).

[0059] 2. The antibacterial activity of the antimicrobial peptide BSN-25 separated from the fusion protein was determined by agar plate diffusion method. The tested microbial strain was Salmonella typhimurium CVCC541 (purchased from China Veterinary Microbiological Culture Collection Center).

[0060] The specific operation steps of the test are as follows: pick a single colony of CVCC 541 into LB medium and culture it at 37°C and 220rpm overnight. Subculture it into 5mL MH liquid medium at a ratio of 1:100 to 0.5 McFarland standard. Use a sterilized cotton swab to evenly smear the above-mentioned test microorganisms on the MH solid culture plate, place a sterile Oxford cup on the culture plate, and add equal volumes of samples, positive control: tetracycline 50μL (30μg / mL), negative control: ddH2O; NZ9000 bacterial solution 100μL; NZ9000 pN8148-M bacterial solution 100μL; synthetic antimicrobial peptide 100μL (100μg / mL); fusion protein enterokinase digestion solution 100μL; let the plate stand for 20-30min to allow the test solution to diffuse into the agar, place it at 37°C for 16-18 hours, and then record the diameter of the inhibition zone around the well. The results are attached. Figure 8 As shown in Table 1 below. From the test graph and the measured inhibition zone diameter, it can be seen that the fusion protein USP 45+BSN-25 has no antibacterial activity, but when it is digested with enterokinase, the digestion fluid shows antibacterial activity, indicating that the antimicrobial peptide BSN-25 has dissociated from the fusion protein and has antibacterial activity.

[0061] Table 1

[0062]

[0063] In summary, the lactic acid genetically engineered bacteria NZ9000 pNZ8148-M:usp45+bsn-25 constructed by the present invention can secrete the fusion protein USP of antimicrobial peptide 45 +BSN-25, no antibacterial activity, the fusion protein can release the antibacterial peptide BSN-25 with antibacterial activity under the action of enterokinase. It shows that the lactic acid genetic engineering bacteria constructed by the present invention can continue to ferment and proliferate in the intestinal environment and exert antibacterial activity, and has broad application prospects in the preparation of antibiotic-free animal health products and antibiotic-free feed additives.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polypeptide for promoting the exocrine expression of a target protein, characterized in that: The amino acid sequence thereof comprises the sequence shown in SEQ ID NO.

1.

2. The polypeptide according to claim 1, characterized in that A secretion enhancing peptide is linked to the carboxyl terminus of the polypeptide according to claim 1.

3. The polypeptide according to claim 2, characterized in that The amino acid sequence of the polypeptide is the sequence shown in SEQ ID NO.

2.

4. A biomaterial, characterized in that: The biological material is a biological material related to the polypeptide according to any one of claims 1 to 3, and is any one of the following 1) to 4): 1) A nucleic acid molecule encoding the polypeptide according to any one of claims 1 to 3; 2) an expression cassette containing the nucleic acid molecule described in 1); 3) a recombinant vector containing the expression cassette described in 2); 4) A recombinant protein expression system comprising 1) the nucleic acid molecule, 2) the expression cassette, or 3) the recombinant vector.

5. The biomaterial according to claim 4, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

3.

6. The biomaterial according to claim 4 or 5, characterized in that: The recombinant protein expression system is a prokaryotic microbial expression system. Preferably, the recombinant protein expression system is a Lactococcus lactis expression system.

7. An application, characterized in that: Use of the polypeptide according to any one of claims 1 to 3 or the biological material according to any one of claims 4 to 6 in preparing a target protein by exocrine expression.

8. The use according to claim 7, characterized in that Use of the polypeptide according to any one of claims 1 to 3 or the biomaterial according to any one of claims 4 to 6 in the preparation of antimicrobial peptides; Or use of the polypeptide according to any one of claims 1 to 3 or the biological material according to any one of claims 4 to 6 in preparing a recombinant Lactococcus lactis that exocrinely expresses an antimicrobial peptide; Or use of the polypeptide according to any one of claims 1 to 3 or the biomaterial according to any one of claims 4 to 6 in the preparation of antimicrobial peptide fusion proteins; Or use of the polypeptide according to any one of claims 1 to 3 or the biological material according to any one of claims 4 to 6 in preparing a recombinant Lactococcus lactis that exocrinely expresses an antimicrobial peptide fusion protein; Or use of the recombinant Lactococcus lactis expressing the antimicrobial peptide fusion protein exocrinely prepared by the polypeptide according to any one of claims 1 to 3 or the biological material according to any one of claims 4 to 6 in the preparation of antibiotic-free animal health products and antibiotic-free feed additives.

9. An antimicrobial peptide fusion protein, characterized in that: The fusion protein is USP 45 A fusion protein obtained by fusing the protein with an antimicrobial peptide for exocrine expression; The exocrine expression adopts the biological material according to any one of claims 4 to 6. Preferably, the USP 45 The protein and antimicrobial peptide are connected by broken peptide chains that can be recognized by enterokinase.

10. A recombinant genetically engineered bacterium, characterized in that: The biomaterial according to any one of claims 4 to 6 is used for exocrine expression of the antimicrobial peptide fusion protein according to claim 9. Preferably, the recombinant genetically engineered bacteria is recombinant Lactococcus lactis.

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