Bacteria-derived antibacterial protein as well as preparation method and application thereof

Abstract

The invention discloses a bacterial source antibacterial protein as well as a preparation method and application thereof. The invention provides an antibacterial protein. The antibacterial protein is prepared by heterologous expression of a bacillus velezensis mrsM gene or protein. The recombinant protein prepared by cloning a bacterial-derived mrsM gene and carrying out heterologous expression in escherichia coli has good antibacterial activity, can inhibit enterobacter aerogenes, also has good stability, can tolerate ultraviolet radiation, has good antibacterial activity at pH of 4-6.5 and below 60 DEG C, and can be used for preparing the recombinant protein for treating the enterobacter aerogenes in the field of animal husbandry, animal husbandry, animal husbandry, animal husbandry, animal husbandry, animal husbandry, animal husbandry, animal husbandry, animal husbandry, animal husbandry and animal husbandry. And the product is not sensitive to papain, trypsin and protease K. Meanwhile, the invention provides a preparation method of the novel antibacterial protein, the preparation method is simple, the source is wide and safe, the antibacterial protein prepared from a bacterial source is provided for prevention and treatment of existing pathogenic edible bacteria, the use safety is better than that of antibiotics, generation of bacterial drug resistance is avoided, and more effective methods and means are provided for prevention and treatment of enterobacter aerogenes.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms and genetic engineering, and more specifically, relates to a bacterial antibacterial protein and a preparation method and application thereof. Background Art

[0002] At present, due to the abuse of antibiotics, more and more bacteria have become more resistant to drugs, and more super-resistant bacteria have emerged, making it difficult to prevent and treat pathogens. Long-term or improper use of antibiotics can cause bacterial gene mutations and form drug-resistant strains. These drug-resistant strains can spread among the population, resulting in a decrease in the effectiveness of conventional antibiotic treatment and even the emergence of "super bacteria", which brings great difficulties to clinical treatment. Due to the emergence of drug-resistant strains, some originally effective antibiotics have become ineffective, increasing the difficulty and cost of treating infectious diseases, and also posing serious challenges to social public health security. Therefore, developing more antibacterial products that can replace the use of antibiotics and expanding the source of antibacterial products are of great significance to the prevention and treatment of pathogens and the development of antibacterial products.

[0003] As concerns about antibiotic resistance grow, more and more studies are beginning to focus on the preparation and development of antimicrobial proteins to find new anti-infection methods. Antimicrobial proteins are biomacromolecules encoded by genes and are an important part of the life defense system. Their main target sites are the components of the cell wall. They interfere with the function of the pathogen cell wall, causing cell growth and cell wall formation to be blocked, or interfere with the structure or function of the cell membrane, thereby inhibiting the growth of pathogens. New antimicrobial proteins have been discovered, such as proline-rich small protein 2A (SPRR2A). These new antimicrobial proteins have unique antimicrobial mechanisms and modes of action, providing new possibilities for anti-infection treatment. Due to the increase in antibiotic resistance, antimicrobial proteins have received widespread attention as potential anti-infection drugs. They have the advantages of high specificity, low toxicity, and low resistance to drug resistance, and are expected to become an important means of anti-infection treatment in the future.

[0004] At present, some heterologous proteins obtained by gene recombination technology also show good antibacterial activity, such as 18kDa cationic antimicrobial peptide (CAMP) recombinant protein, which is obtained by recombinant technology from the protein encoded by the CAMP-18 gene, with characteristics such as polypeptide chain and amino acid sequence, and cationic properties. It can interact with the negative charge on the bacterial cell membrane, destroy the integrity of the bacterial membrane, and then cause bacterial death. At the same time, it can inhibit the formation of bacterial biofilm and enhance the antibacterial effect; and compared with the natural CAMP-18 protein, the recombinant protein has better stability and activity, and is not easily biodegraded. Other antibacterial recombinant proteins such as bacteriocins and defensins also have broad-spectrum antibacterial activity and have stronger killing power against bacteria under certain specific conditions. Compared with antibiotics, antibacterial proteins have many advantages: high specificity, reduced drug resistance, reduced side effects, good stability, reduced drug interactions, etc., which help reduce the risk of treatment failure or adverse reactions caused by drug interactions. These advantages make antibacterial recombinant proteins have broad application prospects and potential in the treatment and prevention of infectious diseases. The sources of antimicrobial proteins are very wide, covering animals, plants, microorganisms, and artificial synthesis. However, there is still a lack of recombinant antimicrobial proteins that can effectively inhibit pathogenic bacteria to replace the use of antibiotics. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies of existing antibacterial proteins and to provide a bacterial-derived antibacterial protein and a preparation method and application thereof.

[0006] The purpose of the present invention is to provide an antibacterial protein.

[0007] Another object of the present invention is to provide a method for preparing the antibacterial protein.

[0008] Another object of the present invention is to provide the application of antibacterial protein.

[0009] Another object of the present invention is to provide an antibacterial product.

[0010] Another object of the present invention is to provide the use of the mrsM gene or protein of Bacillus velez in the preparation of antibacterial proteins.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention provides an antibacterial protein, which is prepared by heterologous expression of the mrsM gene or protein of Bacillus velez. The nucleotide sequence of the mrsM gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein is shown in SEQ ID NO: 2.

[0012] The present invention obtains an mrsM gene in the genome of Bacillus velez through secondary metabolite database analysis and identification, which has a similarity of 100% with mersacidin (lanthipeptide antibiotic) and belongs to Class II lanthipeptide gene; through cloning of the mrsM gene and construction of a recombinant plasmid, heterologous expression is performed in Escherichia coli, and the prepared recombinant protein has good antibacterial activity, can inhibit Enterobacter aerogenes, and also has good stability, has good antibacterial activity between pH 4 and 6.5 and below 60°C, can withstand ultraviolet irradiation, and is insensitive to papain, trypsin, and proteinase K. The present invention uses the bacterial source mrsM gene for heterologous expression in Escherichia coli for the first time, successfully prepares an antibacterial protein, has good antibacterial activity and stability, does not cause pathogenic bacteria to develop drug resistance, and is of great significance for the development of more safe, efficient, and side-effect-free antibacterial products. At the same time, the present invention also provides a method for preparing the antibacterial protein, which is heterologously expressed in Escherichia coli, is simple and convenient, and provides more sources and ways for preparing more antibacterial products that can replace antibiotics.

[0013] The present invention provides a method for preparing the above antimicrobial protein, comprising the following steps: S1. Cloning the mrsM gene and preparing the recombinant plasmid; S2. Transform the recombinant plasmid into Escherichia coli to obtain recombinant bacteria; S3. Perform prokaryotic expression on the recombinant bacteria, collect the bacteria, break them, centrifuge them, and collect the supernatant.

[0014] Preferably, the recombinant plasmid prepared in S1 is prepared using double-enzyme-digested plasmid pET-30a(+).

[0015] Preferably, the primer sequences used to clone the mrsM gene in S1 are shown in SEQ ID NOs: 3-4.

[0016] Preferably, Escherichia coli BL21 (DE3) pLysS competent cells are used in S2.

[0017] Preferably, the prokaryotic expression conditions in S3 are: 0.2-0.6 mM IPTG, 20-40°C, 3-8h.

[0018] In particular, the antibacterial protein obtained after prokaryotic expression is a crude extract, which can be further purified by conventional purification methods and steps in the art. It can be expected that the activity of the purified protein will be improved.

[0019] The present invention provides application of antibacterial protein in inhibiting Enterobacter aerogenes for non-disease diagnosis purposes.

[0020] The present invention provides application of antibacterial protein in preparing antibacterial products.

[0021] The invention provides an antibacterial product containing the antibacterial protein.

[0022] In addition, the present invention also provides the use of the mrsM gene or protein of Bacillus velezinsis in the preparation of antibacterial protein.

[0023] The present invention has the following beneficial effects: The present invention identifies a bacteriocin biosynthetic gene cluster mrsM in Bacillus velez, which has a 100% similarity to mersacidin (lanthipeptide antibiotic), and the gene cluster type belongs to Class II lanthipeptide genes, and its nucleotide sequence is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2. The recombinant protein prepared by cloning the mrsM gene and heterologously expressing it in Escherichia coli has good antibacterial activity, can inhibit Enterobacter aerogenes, and also has good stability, has good antibacterial activity between pH 4 and 6.5 and below 60°C, can withstand ultraviolet irradiation, has high activity, is insensitive to papain, trypsin, and proteinase K, and is only sensitive to pepsin and pronase E. At the same time, a preparation method of the antibacterial protein is also provided. The antibacterial protein can be successfully prepared by heterologous expression in Escherichia coli. The preparation method is simple, convenient, widely available and safe. It provides an antibacterial protein prepared by bacterial-derived genes for the prevention and treatment of existing pathogenic food bacteria. It has better safety than antibiotics, can effectively avoid the development of bacterial resistance, and also provides more effective methods and means for the prevention and treatment of Enterobacter aerogenes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the result of the comparison with the secondary metabolite database antiSMASH (the red one is the mrsM gene cluster).

[0025] Figure 2 1% agarose nucleic acid electrophoresis of the PCR product of mrsM (lane M: DNAmarker5000; lanes 1-10: mrsM).

[0026] Figure 3 1% agarose electrophoresis of double-enzyme-digested plasmid (lane M: DNA marker 15000; lane 1: undigested plasmid PET-30a(+); lanes 2-7: double-enzyme-digested plasmid PET-30a(+)).

[0027] Figure 4 Agarose gel electrophoresis of colony PCR products (lane M: DNAmarker5000; lane 1: blank control; lane 2: mrsM colony PCR).

[0028] Figure 5SDS-PAGE of the target protein (M represents Marker130; 1 represents the supernatant of BL21 transformed with the recombinant plasmid; 2 represents the precipitate of BL21 transformed with the recombinant plasmid; 3 represents the supernatant of BL21 without the empty plasmid; 4 represents the precipitate of BL21 transformed with the empty plasmid).

[0029] Figure 6 For the antibacterial activity determination of the expressed protein (1 represents the blank control; 2 represents the antibacterial activity of the untransformed Escherichia coli BL21 against Enterobacter aerogenes; 3 represents the antibacterial activity of the transformed Escherichia coli BL21 against Enterobacter aerogenes).

[0030] Figure 7 Optimization of bacteriocin induction conditions ( Figure 7 A in is the temperature; Figure 7 B in it is time; Figure 7 The C in the figure is IPTG). DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0032] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0033] The culture medium formula used in the following examples is: LB broth: 10 g / L tryptone, 5.0 g / L yeast powder, 10.0 g / L sodium chloride.

[0034] LB agar: 10 g / L tryptone, 5.0 g / L yeast powder, 10.0 g / L sodium chloride, 15.0 g / L agar.

[0035] Fermentation medium: 4.5 g / 100 mL glucose, 1.5 g / 100 mL yeast powder, 1.2 g / 100 mL magnesium sulfate heptahydrate.

[0036] SOC liquid medium: 20.0 g / mL tryptone, 5.0 g / mL yeast extract, 0.5 g / mL NaCl, 0.186 g / mL KCl, 0.95 g / mL MgCl 2 、1.2 g / mL MgSO 4 , 3.6 g / mL glucose.

[0037] Example 1 Identification and cloning of bacterial mrsM genes 1. Identification of bacterial mrsM genes Based on the results of the whole genome sequencing of Bacillus velez, a prediction analysis of known secondary metabolic gene clusters was performed in the antiSMASH database. Figure 1 As shown, a bacteriocin biosynthesis gene cluster mrsM was identified in the genome of Bacillus Velezii through the secondary metabolite database antiSMASH, with a similarity of 100% to mersacidin (lanthipeptide antibiotic). The gene cluster type was identified as class II lanthipeptide genes. The nucleotide sequence of the gene cluster mrsM is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.

[0038] 2. Cloning of mrsM gene fragment (1) Acquisition of target genes The Bacillus Velezii stored at -80°C was inoculated into 5 mL of LB broth and then inoculated into 50 mL of fermentation medium at a 1% inoculum. The culture was shaken at 30°C and 180 rpm for 24 h. DNA was extracted from the obtained bacteria according to the instructions of the Bacterial DNA Kit (OMEGA, USA) and set aside.

[0039] (2) Primer design The mrsM gene sequence SEQ ID NO: 1 obtained by the above identification was used to design primers using the online primer design software CE Design (http: / / www.vazyme.com) on the website of the biological company. The designed and synthesized primers are as follows: F(SEQ ID NO:3)5'-gccatggctgatatc ggatcc ATGCATACAAAATTCAAACGGAAT-3' (the underline indicates the BamHI restriction site of the homologous sequence at the end of the upstream vector); R(SEQ ID NO:4)5'-gtggtggtggtggtg ctcgag TTACAATTTTAGTTCTAAAGGTGATTGG-3' (the underline indicates the XhoI restriction site of the downstream vector end homologous sequence).

[0040] (3) Amplification of bacteriocin genes The extracted genomic DNA of Bacillus velez was used as a template and the designed upstream and downstream primers were used to amplify the target gene. The reaction system and reaction conditions for the PCR reaction are shown in Tables 1 and 2 below.

[0041] Table 1 PCR reaction system

[0042] Table 2 PCR reaction conditions

[0043] The target band of the PCR product was detected by 1% agarose gel electrophoresis, and then the target band was recovered and purified using a common agarose gel DNA recovery kit (TIANGEN) (stored at -20°C), and the concentration was measured by spectrophotometer.

[0044] Gel electrophoresis results Figure 2 As shown, the size of the amplified target product is consistent with the actual size, which is 3189 bp, with a single band and no non-specific bands. The band was recovered and the concentration was measured by spectrophotometer to be 46.5 ng / μL, and the mrsM gene fragment was obtained.

[0045] Example 2 Construction of E. coli expression system 1. Construction of recombinant plasmid (1) Plasmid extraction The competent cells containing the PET-30a(+) empty plasmid stored at -80°C were streaked onto four zones of LB solid agar plates containing 25 μg / mL kanamycin and cultured upside down at 37°C overnight. A single colony was picked and inoculated into 50 mL LB broth medium containing 25 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm. The plasmid was extracted using a plasmid mini-extraction reagent (TIANGEN).

[0046] (2) Double enzyme digestion recovery The pET-30a(+) empty plasmid was double-digested, and the double-digestion system is shown in Table 3 below.

[0047] Table 3 Double restriction enzyme digestion system of pET-30a(+)

[0048] The empty plasmid was double-digested with BamHI and XhoI restriction endonucleases at 37 °C for 30 min. After the digestion was completed, the double-digestion effect of the system was verified by 1% (W / V) agarose gel electrophoresis, and the target band was cut and recovered and purified using a common agarose gel DNA recovery kit (TIANGEN), and the concentration was measured by spectrophotometer.

[0049] Test results such as Figure 3 As shown, it can be seen that the size of the pET-30a(+) plasmid is consistent with the theoretical value (5236 bp), and the band is single and clean, without chromosomal DNA breakage contamination. The band was cut and recovered by gel recovery kit, and the concentration was determined by spectrophotometer to be 98.7 ng / μL.

[0050] (3) Homologous recombination The double-enzyme-cut plasmid and the insert fragment (the mrsM gene fragment of Example 1) were homologously recombined at a molar ratio of 1:1. The homologous recombination reaction system is shown in Table 4 below. The reaction system was cooled to 4°C at 50°C for 30 min or immediately placed on ice to obtain a recombinant plasmid product. The amount of the insert fragment was calculated as follows: Optimal amount of cloning vector used = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol) Optimal amount of insert fragment = [0.02 × number of base pairs of insert fragment] ng (0.03 pmol) Table 4 Homologous recombination reaction system

[0051] 2. Transformation of recombinant plasmid (1) Conversion Thaw the DH5α chemical competent cells on ice, add the above recombinant product to 100 μL competent cells, flick the tube wall to mix (do not oscillate to mix), and let stand on ice for 30 min. Heat shock in a 42°C water bath for 30 sec, and immediately cool on ice for 2-3 min. Add 900 μL SOC liquid medium (without antibiotics), and shake at 37°C for 1 h (speed 220rpm). Centrifuge at 5000 rpm for 5 min, and discard 900 μL supernatant. Resuspend the bacteria with the remaining culture medium, gently spread on the LB agar plate containing 50 mg / mL kanamycin with a sterile applicator, and invert and culture overnight in a 37°C incubator. Transform the recombinant plasmid into E. coli competent cells. E. coli carrying the successfully transformed recombinant plasmid has kanamycin resistance and can survive on the LB solid plate containing kanamycin.

[0052] (2) Screening and identification - colony PCR Pick a single colony from the plate and add it to 40 μL ddHO. 2 O and mix thoroughly. Take 10 μL and send it to Bio-Tech for sequencing, and take 3 μL for colony PCR detection. The PCR system is shown in Table 5.

[0053] Table 5 PCR reaction system

[0054] Then, 1% agarose gel electrophoresis was used to detect the target band of the PCR product.

[0055] Agarose gel electrophoresis results Figure 4 As shown, the agarose gel electrophoresis of the colony PCR product is consistent with the actual size. Combined with the sequencing comparison analysis results, it is proved that the expression vector was successfully constructed.

[0056] Example 3 Inducible expression of recombinant protein 1. Transfer vector The identified positive recombinant plasmid was inoculated into LB broth. The next day, the recombinant plasmid was extracted according to the instructions of the plasmid extraction kit, and 10 μL of the recombinant plasmid was transformed into 50 μL of Escherichia coli BL21 (DE3) pLysS competent cells. The transformation method was the same as in Example 2 to obtain recombinant bacteria.

[0057] 2. Inducible expression The recombinant bacteria after successful transformation were inoculated into 100 mL of LB liquid medium containing 0.4 mg / mL kanamycin and cultured in a constant temperature shaker at 37°C at 100 rpm until OD 600 nm was 0.6; then IPTG with a final concentration of 0.5 mM was added and induced at 37°C 220 rpm for 5 h. The bacterial solution was then centrifuged at 4°C and 4000 rpm for 20 min to collect the cells, and the cells were washed three times with pH 7.0 PBS buffer to remove residual kanamycin, and then resuspended in pH 7.0 PBS buffer; then a cell disruptor was used to completely disrupt the cells at 60% intensity, 5s disruption, 7s, 4 min one cycle, repeated three times, for a total of 12 min. Finally, the bacterial solution was centrifuged at 4°C and 12000 rpm for 20 min, and the supernatant and precipitate were collected for protein SDS-PAGE detection and verification.

[0058] The SDS-PAGE results of the target protein are as follows: Figure 5 As shown, after 4 h of IPTG induction, a new induced band appeared at around 121.1 kDa in the recombinant bacteria BL21 (DE3), which was consistent with the expected protein size (121.1 kDa); some recombinant bacteria transformed with PET-30(+) empty plasmid did not show this protein band, indicating that the mrsM protein was successfully expressed in E. coli.

[0059] 3. Antibacterial activity The antibacterial activity was determined by the agar punch method, and the indicator bacteria used were Enterobacter aerogenes, Listeria monocytogenes, and Escherichia coli. The indicator bacteria were inoculated into 5 mL LB broth at 2% and cultured overnight at 37°C; the obtained seed liquid was inoculated into 30 mL LB agar at 1%, poured into the plate, and after solidification, a sterile punch was used to punch. Then the above recombinant protein was filtered through a 0.22 μm filter membrane, added to the well, and cultured overnight at 37°C.

[0060] The test results showed that the recombinant protein had significant antibacterial activity and could significantly inhibit Enterobacter aerogenes, but had no effect on other indicator bacteria (Listeria monocytogenes and Escherichia coli). The results of inhibition on Enterobacter aerogenes were as follows: Figure 6As shown, the diameter of the inhibition zone against Enterobacter aerogenes was 14.40 mm.

[0061] Example 4 Optimization of expression conditions of antimicrobial proteins The recombinant bacteria BL21 (DE3) in Example 3 were inoculated into 100 mL of LB liquid culture medium containing 50 μl / L kanamycin, and cultured overnight at 37°C and 220 rpm to obtain seed bacteria. The following experiments were performed respectively: (1) Induction temperature optimization Add kanamycin to a final concentration of 50 mg / mL to each of the six 100 mL LB liquid media, and then add 1% (V / V) seed solution to each bottle. Incubate at 37°C, 220 rpm in a shaking incubator until the OD 600 to 0.6, respectively, and add 0.5 mM IPTG to all culture media, and culture at 25, 30, and 37°C, 130 rpm for 5 h. The remaining steps of inducing expression are the same as those in Example 3, and finally, the expression protein samples at different culture times are obtained, and then the agar punch method in Example 3 is used for verification.

[0062] Test results such as Figure 7 As shown in A, the expression conditions of the protein were optimized, showing that the antibacterial activity of the recombinant protein was highest when the induction temperature was 30°C.

[0063] (2) Optimization of induction time Add kanamycin to a final concentration of 50 mg / mL to each of the six 100 mL LB liquid media, and then add 1% (V / V) seed solution to each bottle. Incubate at 37°C, 220 rpm in a shaking incubator until the OD 600 When the pH value reached 0.6, 0.5 mM IPTG was added to all the culture media, and the culture was carried out at 30°C and 130 rpm for 4, 5, 6, and 7 h, respectively. The remaining steps of inducing expression were the same as those in Example 3, and finally, the expression protein samples at different culture times were obtained, and then the agar punching method in Example 3 was used for verification.

[0064] Test results such as Figure 7 As shown in B, the protein expression conditions were optimized, showing that the antibacterial activity of the recombinant protein at an induction time of 5 h was the highest.

[0065] (3) IPTG optimization Add kanamycin at a final concentration of 50 mg / mL to each of the six 100 mL LB liquid cultures, and then add 1% (V / V) seed solution to each bottle. Incubate at 37°C, 220 rpm in a shaking incubator until the OD 600to 0.6, respectively, and 0.3, 0.4, 0.5, and 0.6 mM IPTG were added to all the culture media, respectively, and cultured at 30°C, 220 rpm for 5 h. The remaining steps of inducing expression were the same as those in Example 3, and finally, the expression protein samples at different culture times were obtained, and then verified by the agar punching method in Example 3.

[0066] Test results such as Figure 7 As shown in C, the protein expression conditions were optimized, showing that the antibacterial activity of the recombinant protein was highest when the IPTG concentration was 0.4 mM.

[0067] Example 5 Determination of the stability performance of antimicrobial proteins (1) pH stability The recombinant protein prepared in Example 3 was divided into five portions, each with 1 mL. The pH values ​​were adjusted to 2, 4, 8, and 10 respectively with 1 mol / L NaOH and 1 mol / L HCl, and the pH was adjusted back to neutral again after being placed at 4°C for 12 h. The agar punch method in Example 3 was then used for verification, and the pH stability of the recombinant protein was determined by repeating three groups.

[0068] The results are shown in Table 6, which show that the recombinant protein has antibacterial activity between pH 4 and 6.5, and has no antibacterial effect and activity when the pH reaches 8.

[0069] Table 6 pH stability of recombinant protein

[0070] Note: - indicates no antibacterial effect; compared with the control group: * indicates significant difference ( P <0.05, ** indicates extremely significant difference ( P <0.01).

[0071] (2) Temperature stability The recombinant protein prepared in Example 3 was divided into five portions, each with 1 mL, and treated in a water bath at 40°C, 60°C, 80°C, and 100°C for 30 min, and in a high pressure treatment at 121°C for 20 min. Then, the agar punching method in Example 3 was used for verification, and three groups were repeated to determine the temperature stability of the recombinant protein.

[0072] The results are shown in Table 7, which show that the recombinant protein has antibacterial activity at temperatures between 40 and 60°C, maintains antibacterial activity below 60°C, and disappears as the temperature rises. When the temperature reaches 80°C, there is no antibacterial effect and no activity.

[0073] Table 7 Temperature stability of recombinant proteins

[0074] Note: - indicates no antibacterial effect; compared with the control group: * indicates significant difference ( P <0.05, ** indicates extremely significant difference ( P <0.01).

[0075] (3) UV stability The recombinant protein prepared in Example 3 was divided into five portions, each with 1 mL. They were placed under a 15W UV lamp at a distance of about 15 cm and irradiated for 10 min, 20 min, 40 min, 60 min, and 90 min, respectively. The agar punch method in Example 3 was then used for verification, and three groups were repeated to determine the UV stability of the recombinant protein.

[0076] The results are shown in Table 8, which show that the recombinant protein still has antibacterial activity after UV irradiation for 10 to 90 minutes. Under UV irradiation for different times, the activity remains stable, and the antibacterial activity decreases significantly after 60 minutes.

[0077] Table 8 UV stability of recombinant proteins

[0078] Note: - indicates no antibacterial effect; compared with the control group: * indicates significant difference ( P <0.05, ** indicates extremely significant difference ( P <0.01).

[0079] (4) Enzyme stability The recombinant protein prepared in Example 3 was divided into five portions, each with 1 mL. Pepsin, trypsin, papain, proteinase K, and pronase E (1 g / mL) were added respectively, and heated in a water bath at 37°C for 1 hour. Then, the agar punching method in Example 3 was used for verification, and three groups were repeated to determine the enzyme stability of the recombinant protein.

[0080] The results are shown in Table 9, which show that the recombinant protein still has antibacterial activity in the enzymatic hydrolysis environments of papain, trypsin, and proteinase K, while the recombinant protein is most sensitive to pepsin and pronase E, and the activity disappears.

[0081] Table 9 Recombinant protease stability

[0082] Note: - indicates no antibacterial effect; compared with the control group: * indicates significant difference ( P <0.05, ** indicates extremely significant difference ( P <0.01).

[0083] In summary, the present invention identified a bacteriocin biosynthetic gene cluster mrsM in Bacillus velez, which has a 100% similarity to mersacidin (lanthipeptide antibiotic). The gene cluster type belongs to Class II lanthipeptides, and its nucleotide sequence is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2. By cloning the mrsM gene and heterologously expressing it in Escherichia coli, the recombinant protein prepared has good antibacterial activity, can inhibit Enterobacter aerogenes, and provides a source for the preparation of more antibacterial proteins; at the same time, the recombinant protein also has good stability, has good antibacterial activity between pH 4 and 6.5 and below 60°C, can withstand ultraviolet irradiation, is insensitive to papain, trypsin, and proteinase K, and has high activity. The present invention also provides a method for preparing bacterial-derived antimicrobial proteins. The preparation method has simple and convenient steps, a wide range of sources, and is safe. It provides antimicrobial proteins prepared from bacterial sources for the prevention and treatment of existing pathogenic food bacteria. It has better safety than antibiotics, avoids the generation of bacterial resistance, and also provides more effective methods and means for the prevention and treatment of Enterobacter aerogenes.

[0084] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An antimicrobial protein, characterized in that The protein is prepared by heterologously expressing the mrsM gene or protein of Bacillus velez. The nucleotide sequence of the mrsM gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein is shown in SEQ ID NO:

2.

2. The method for preparing the antimicrobial protein according to claim 1, characterized in that: The following steps are involved: S1. Cloning the mrsM gene according to claim 1 to prepare a recombinant plasmid; S2. Transform the recombinant plasmid into Escherichia coli to obtain recombinant bacteria; S3. Perform prokaryotic expression on the recombinant bacteria, collect the bacteria, break them, centrifuge them, and collect the supernatant.

3. The method according to claim 2, characterized in that: The recombinant plasmid in S1 was prepared by double-enzyme digestion of plasmid pET-30a(+).

4. The method according to claim 2, characterized in that: The primer sequences used in S1 to clone the mrsM gene described in claim 1 are shown in SEQ ID NOs: 3~4.

5. The method according to claim 2, characterized in that: In S2, E. coli BL21(DE3) pLysS competent cells were used.

6. The method according to claim 2, characterized in that: The prokaryotic expression conditions in S3 were: 0.2-0.6 mM IPTG, 20-40°C, 3-8 h.

7. Use of the antimicrobial protein according to claim 1 in inhibiting Enterobacter aerogenes for non-disease diagnosis purposes.

8. Use of the antibacterial protein according to claim 1 in the preparation of antibacterial products.

9. An antibacterial product, characterized in that: Containing the antibacterial protein according to claim 1.

10. Use of the mrsM gene or protein of Bacillus velez in the preparation of antibacterial protein, characterized in that: The nucleotide sequence of the mrsM gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein is shown in SEQ ID NO: 2.

Images