Bacteriocin expressed by bacillus subtilis wb800n and application thereof

Abstract

The application discloses a bacteriocin expressed by bacillus subtilis WB800N and application thereof. The original sequence of the bacteriocin is a bacteriocin PA from bacillus polymyxa, the nucleotide sequence of the bacteriocin PA is shown as SEQ ID NO:1, and the amino acid sequence is shown as SEQ ID NO:2. The bacteriocin PA coding gene is modified by a tag and optimized by a codon to obtain a bacteriocin PFH, the nucleotide sequence of the bacteriocin PFH is shown as SEQ ID NO:3, and the amino acid sequence is shown as SEQ ID NO:4. The bacteriocin PFH expressed by the bacillus subtilis WB800N has a good bacteriostatic effect on gram-positive bacteria such as streptococcus suis, enterococcus faecium, staphylococcus aureus and bacillus subtilis, and has a wide application prospect in the development of a new type of replacement product for infection treatment and a feed antibacterial peptide.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and biotechnology, specifically to a bacteriocin expressed by Bacillus subtilis WB800N and its application. Background Technology

[0002] Since the 20th century, antibiotics have played a vital role in preventing and treating human bacterial infections, as well as in the prevention and treatment of animal diseases, improving feed utilization efficiency, and promoting rapid growth in livestock and poultry. However, with the long-term use or abuse of antibiotics, the problem of bacterial resistance has become increasingly serious. Therefore, the development of novel, highly effective, and safe antimicrobial drugs to address the dual pressures of severe pathogenic infections and pathogenic microbial resistance is particularly urgent. Certain naturally synthesized antimicrobial peptides by microorganisms have shown great potential as alternatives. Antimicrobial peptides are small, bioactive polypeptides induced in organisms, with molecular weights ranging from 2000 to 7000, composed of 20 to 60 amino acid residues. Most of these active polypeptides possess advantages such as strong alkalinity, thermal stability, antibacterial activity, low resistance to drug development, and no residue.

[0003] Bacillus is a group of facultative anaerobic or aerobic bacteria, a general term for Gram-positive bacilli. They produce spores, exhibit diverse physiological characteristics, and are widely distributed on plant surfaces and roots, in soil, and in the air. Numerous studies have shown that Bacillus can produce a variety of active substances with broad antibacterial spectra, most of which are polypeptides. The active peptides produced by Bacillus have inhibitory activities against fungi such as molds, Gram-positive bacteria, and Gram-negative bacteria; some also possess antiviral and anti-mycoplasma properties. Based on their chemical structure, antimicrobial peptides produced by Bacillus are mainly classified into bacteriocins, glycopeptides, lipopeptides, and cyclic peptides.

[0004] Currently, the commonly used prokaryotic expression system for protein expression is Escherichia coli. Its advantages include mature technology, short expression cycle, and high expression levels. However, it also has some disadvantages: 1. The expressed protein has not been modified and cannot be guaranteed to have the activity of the natural protein. The expressed protein can be used for antibody preparation or detection, but is rarely used to express biologically active proteins. 2. It is impossible to regulate the expression time and level. Overexpression of some target genes can lead to non-physiological responses. Target proteins are often expressed in the form of inclusion bodies, making product purification difficult.

[0005] Although there have been many studies on the expression of antimicrobial peptides in Escherichia coli and Bacillus subtilis expression systems, there are currently no reports on the exogenous expression of the potential bacteriocinogen gene PFH predicted from Bacillus polymyxa. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a bacteriocin expressed by Bacillus subtilis WB800N and its application. The polymyxa-derived bacteriocin PA predicted by this invention is derived from Bacillus polymyxa (…). Paenibacillus polymyxa This invention relates to a RiPP precursor polypeptide, a member of the NHLP leader peptide family, predicted in the whole genome of *Bacillus polymyxa*. The *P. polymyxa*-derived bacteriocin PA of this invention, after tag modification and codon optimization, yielded the antibacterial recombinant bacteriocin PFH. Its physicochemical properties and antibacterial activity were investigated through heterologous expression in *Bacillus subtilis* WB800N, and its potential for application as an alternative antibiotic for infection treatment and as a feed additive for animals was preliminarily explored.

[0007] To achieve the above objectives, the technical solution designed by the present invention is as follows: The present invention provides a bacteriocin PA, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0008] The present invention also provides a nucleotide sequence encoding bacteriocin PA as shown in SEQ ID NO: 1.

[0009] This invention also provides a method for constructing recombinant Bacillus subtilis WB800N, comprising the following steps: (1) The nucleotide sequence of bacteriocin PA was obtained by the online bacteriocin prediction website BAGEL4. The 5′ end of the nucleotide sequence was linked to the ribosome binding site RBS, and the 3′ end was linked to the 3×flag and 6×His tags respectively. After codon optimization, the antibacterial recombinant bacteriocin gene PFH was obtained. (2) The antibacterial recombinant bacteriocin gene PFH was ligated into the pHT43 vector. BamH I and Sma Between the restriction enzyme sites, the plasmid was transferred into E. coli, and the plasmid was extracted to obtain the pHT43-PFH recombinant plasmid. (3) The pHT43-PFH plasmid was electroporated into Bacillus subtilis WB800N, and recombinant Bacillus subtilis WB800N was obtained after screening.

[0010] Further, in step (1), the nucleotide sequence of the antimicrobial recombinant bacteriocin gene is shown in SEQ ID NO: 3; the amino acid sequence of the antimicrobial recombinant bacteriocin gene is shown in SEQ ID NO: 4.

[0011] This invention also provides a method for preparing antibacterial recombinant bacteriocin PFH, comprising the following steps: The recombinant Bacillus subtilis obtained by the above construction method was streaked on WB800N plates and cultured. Single colonies were picked and placed in LB broth for activation. The activated bacterial solution was transferred into LB broth and cultured with shaking to expand the culture. Isopropyl-β-D-thiogalactoside was added to induce expression. The fermentation supernatant was collected by centrifugation. The fermentation supernatant was the antibacterial recombinant bacteriocinol PFH.

[0012] Furthermore, the culture and activation conditions are 37°C, 220 rpm, and 8–12 h. The activated bacterial solution was transferred into LB broth at a volume ratio of 1:100, and cultured with shaking at 37°C and 220 rpm until the bacterial solution reached its OD value. 600 To 0.8~1.2; Isopropyl-β-D-thiogalactoside was added to a final concentration of 0.1–2 mmol / L, and the induction conditions were 28–32 °C, 200 rpm, and 48–72 h.

[0013] The present invention also provides the application of the antibacterial recombinant bacteriocin PFH prepared by the preparation method described above in inhibiting bacterial activity, wherein the bacteria are any one of Staphylococcus aureus, Micrococcus luteus, Enterococcus faecalis, Streptococcus suis, Bacillus subtilis, and Bacillus polymyxa.

[0014] The present invention also provides the application of the antibacterial recombinant bacteriocin PFH prepared by the preparation method described above in the preparation of antibacterial agents.

[0015] The present invention also provides an antibacterial agent comprising the antibacterial recombinant bacteriocin PFH prepared by the preparation method described above, wherein the concentration of the antibacterial recombinant bacteriocin PFH in the antibacterial agent is 8~10 μg / mL.

[0016] The present invention also provides the application of the antibacterial agent in inhibiting bacterial activity, wherein the bacteria are any one of Staphylococcus aureus, Enterococcus faecium, and Streptococcus suis.

[0017] The beneficial effects of this invention are: 1. Bacillus subtilis WB800N prokaryotic expression of Bacillus polymyxa-derived antibacterial recombinant PFH against G + The antibacterial effect against Staphylococcus aureus (a Gram-positive bacterium) is good, indicating that the Bacillus subtilis WB800N expression system was successfully constructed and is suitable for the expression of short peptides while maintaining their antibacterial activity. The expression of antibacterial recombinant bacteriocin PFH using this system has great application prospects in wound infection treatment and feed additives.

[0018] 2. This invention designs and expresses an antimicrobial recombinant bacteriocin PFH based on the predicted Bacillus polymyxa-derived bacteriocin PA. After prokaryotic expression in Bacillus subtilis WB800N, activity assays revealed that the antimicrobial recombinant bacteriocin PFH exhibits antibacterial effects. Furthermore, the preparation process is simple and convenient for formulation applications (e.g., high-activity protein can be isolated simply by removing bacteria from the fermentation broth). The antimicrobial recombinant bacteriocin PFH obtained by this invention has broad application prospects in the development of novel antibiotic alternatives and antimicrobial peptides for livestock and poultry feed.

[0019] 3. The addition of a His tag to the carboxyl terminus of the polymyxa-derived recombinant antibacterial bacteriocin PFH of this invention makes the bacteriocin easier to detect, identify, and purify. Antimicrobial testing results confirm that the recombinant antimicrobial bacteriocin PFH with excellent antimicrobial properties can be obtained using the Bacillus subtilis expression system (specifically, the Bacillus subtilis WB800N expression strain), and can be used as a relatively stable exogenous antimicrobial agent in livestock and poultry feed additives. Attached Figure Description

[0020] Figure 1 PCR validation of the vector's pHT43 double digestion linearization; Figure 2 The image shows the electrophoresis results of the target gene fragment PFH. Figure 3 This is a diagram showing the PCR verification results of E. coli colonies. Figure 4 This is a colony morphology diagram of Bacillus subtilis WB800N; Figure 5 Bacillus subtilis WB800N OD 600 The effect of conversion rate is shown in the figure. Figure 6 Colony plate image for screening recombinant Bacillus subtilis WB800N; Figure 7 The image shows the PCR results of recombinant Bacillus subtilis WB800N bacterial culture. Figure 8 Western blot analysis of the purification effect of antibacterial recombinant bacteriocin PFH; In the figure, M: marker; FS: fermentation supernatant; FT: flow through; W1~W3: wash 1~3; E1~E4: elution 1~4. Figure 9 For the determination of protein concentration in fermentation products using the Bradford method; Figure A shows the Oxford Cup drug susceptibility test: the drug susceptibility results of recombinant Bacillus subtilis WB800N / pHT43-PFH obtained by fermentation at 30℃ for different times to obtain the antibacterial recombinant bacteriocin PFH; Figure B shows the effect of different induction times on the expression level of antibacterial recombinant bacteriocin PFH. Figure 10 Growth curves of recombinant Bacillus subtilis WB800N / pHT43 and recombinant Bacillus subtilis WB800N / pHT43-PFH; Figure 11 Figure showing the effect of the optimal IPTG induction concentration on the expression level of recombinant bacteriocin PFH. Figure 12 The effect of optimal induction timing on the expression level of recombinant antibacterial bacteriocin PFH (Figure 1). Figure 13 Figure 1 shows the growth and antibacterial activity secretion of recombinant Bacillus subtilis WB800N / pHT43-PFH under different induction times. Figure 14 Figure 1 shows the effect of different pH culture media on the growth of recombinant Bacillus subtilis WB800N / pHT43-PFH and the antibacterial activity of its antibacterial active substances. Figure 15 Figure 1 shows the effect of adding different proportions of lactose to the basal culture medium on the growth of recombinant Bacillus subtilis WB800N / pHT43-PFH and the antibacterial activity of its antibacterial active substances. Figure 16 The graph shows the thermal stability of the antibacterial recombinant bacteriocin PFH. Figure 17 The graph shows the pH stability of the antibacterial recombinant bacteriocin PFH. Figure 18 Figure showing the results of a stability study of the antibacterial recombinant bacteriocin PFH against proteases (>1000 U / mL); Figure 19 Figure showing the results of a study on the stability of the antibacterial recombinant bacteriocin PFH against UV radiation; Figure 20 Figure showing the results of a study on the stability of recombinant bacteriocin PFH against metal salt ions at physiological concentrations; Figure 21 Figure showing the results of a stability study of the antibacterial recombinant bacteriocin PFH on surfactants; Figure 22 This is a graph showing the antibacterial activity results of the antibacterial agent; In the figure, a~c are the quality control bacteria Escherichia coli ATCC-25922, where a: Bacillus subtilis WB800N (fermentation broth 2); b: recombinant Bacillus subtilis WB800N / pHT43 (fermentation broth 1); c: recombinant Bacillus subtilis WB800N / pHT43-PFH (antibacterial agent). d~f are the quality control bacteria Staphylococcus aureus ATCC-29213, where d: Bacillus subtilis WB800N (fermentation broth 2); e: recombinant Bacillus subtilis WB800N / pHT43 (fermentation broth 1); f: recombinant Bacillus subtilis WB800N / pHT43-PFH (antibacterial agent). Figure 23 This is a graph showing the results of an antibacterial spectrum study of antibacterial agents; In the figure, the test strains are a: Micrococcus luteus; b: Staphylococcus aureus ATCC-29213; c: Enterococcus faecium ATCC JH2-2; d: Streptococcus suis ATCC 43765. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0022] Example 1 Construction of recombinant plasmid pHT43-PFH 1. Obtaining fragments for potential bacteriocins Based on the gene sequence information of Bacillus polymyxa bxy whole genome sequencing, the potential bacteriocin encoding gene PA was obtained by predicting BAGEL4 through a bacteriocin online prediction website. Its nucleotide sequence is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2.

[0023] 2. Vector linearization by double digestion with pHT43 use BamH Ⅰ and Sma Ⅰ The pHT43 vector was double-digested to obtain a linearized vector. The double digestion reaction system is shown in Table 1.

[0024] Table 1. Double enzyme digestion reaction system Enzyme digestion conditions: reaction at 37℃ for 1 h. The digestion products were detected by 1% agarose gel electrophoresis. Electrophoresis conditions: 150V voltage, 15 min, with DL 2000 DNA Marker as a reference. Exposure and photography were recorded using a gel UV imaging system.

[0025] The results of electrophoresis detection of the obtained double enzyme digestion products are as follows: Figure 1As shown, the nucleic acid electrophoresis position of the linearized pHT43 vector is larger than that of the circular pHT43 vector, indicating that the pHT43 vector is completely linearized by double enzyme digestion. The corresponding band size of the linearized pHT43 gene is 8057bp.

[0026] 3. Obtain the target gene fragment PFH (1) The 5′ end of the bacteriocin gene PA was linked to the ribosome binding site RBS, and the 3′ end was linked to the 3×flag and 6×His tags in sequence. After codon optimization, the antibacterial recombinant bacteriocin gene PFH was obtained. Its nucleotide sequence is shown in SEQ ID NO: 3, and the amino acid sequence of the antibacterial recombinant bacteriocin gene is shown in SEQ ID NO: 4.

[0027] (2) Design fusion primers (PFH-fusion-F and PFH-fusion-R) to amplify the PFH gene; the nucleotide sequences of the fusion primers are shown in Table 2.

[0028] Table 2 PCR amplification primers and identification primers By designing fusion primers, the PFH gene fragment was obtained by PCR amplification from a recombinant plasmid synthesized by Beijing Qingke Biotechnology Co., Ltd. The results of electrophoresis analysis of the PCR products are shown below. Figure 2 As shown, the band size corresponding to the target gene PFH fragment is 342 bp, which is consistent with the expectation.

[0029] 4. Construction and validation of recombinant plasmid pHT43-PFH (1) Homologous recombination of the target gene PFH and the vector pHT43 The recombinant plasmid pHT43-PFH was constructed using the homologous recombinase 2×MultiF Seamless Assembly Mix (ABclonal), and the antibacterial recombinant bacteriocin gene was ligated into the pHT43 vector. BamH I and Sma Between the restriction enzyme sites. The homologous recombination system is shown in Table 3. Incubation at 50℃ for 60 min yielded the homologous recombination product.

[0030] Table 3 Homologous recombination system (2) Transformation of recombinant plasmids into competent Escherichia coli cells DH5α Remove competent DH5α E. coli cells from -80°C and thaw them rapidly on ice. Add the homologous recombinant product to 100 μL of competent cells, gently tap the tube to mix, and incubate on ice for 30 min. After heat shock in a 42°C water bath for 90 s, immediately incubate on ice for 2 min, without shaking the centrifuge tube. Add 900 μL of LB or SOC liquid medium (antibiotic-free) to the centrifuge tube, mix well, and incubate at 37°C and 200 rpm for 1 h. Centrifuge at 5000 rpm for 3 min, discard 900 μL of supernatant, resuspend the bacterial culture in the remaining medium, and spread evenly on LB agar plates containing 100 μg / mL ampicillin. Incubate the plates upright at 37°C for 10 min until the bacterial culture is completely absorbed, then invert the plates and incubate overnight at 37°C to grow E. coli colonies.

[0031] (3) PCR verification of Escherichia coli colonies PCR verification of E. coli colonies was performed, and the colony PCR results are as follows: Figure 3 As shown, the positive control is the target fragment PFH, with a sequence length of 342 bp. The fragment length of the plasmid pHT43-PFH amplified using pHT43-F / pHT43-R primers is 775 bp. The colony PCR results are consistent with the above fragment length, indicating that the pHT43-PFH recombinant plasmid was successfully constructed.

[0032] (4) Extraction of recombinant plasmids Positive Escherichia coli colonies were picked and inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin). The strains were activated by culturing at 37°C and 220 rpm for 6 h. The activated strains were then transferred to 20 mL of LB broth at a ratio of 1:1000 and cultured at 37°C and 220 rpm for 12–16 h. Recombinant plasmids were extracted using a plasmid extraction kit.

[0033] (5) Identification of recombinant plasmids The extracted recombinant plasmid was amplified by PCR using the verification primers pHT43-F / pHT43-R. The PCR amplification products were detected by 1% agarose gel electrophoresis. The nucleotide sequences of primers pHT43-F and pHT43-R are shown in Table 2.

[0034] (6) Recombinant plasmid sequence analysis The recombinant plasmid was sent to the company for sequencing. The sequence obtained by sequencing was compared with the antibacterial recombinant protein gene sequence using Snapgene software. The sequencing results were consistent with the antibacterial recombinant protein gene PFH sequence, indicating that the recombinant plasmid pHT43-PFH was successfully constructed.

[0035] Example 2: Prokaryotic expression of recombinant antibacterial bacteriocin PFH in Bacillus subtilis WB800N 1. Identification of Bacillus subtilis WB800N strain Bacillus subtilis WB800N glycerol culture (purchased from Beijing Zoman International Biotechnology Co., Ltd.; http: / / www.zomanbio.com / shengwu_info.php?nid=1742), stored at -80℃, was activated overnight at 37℃ and 220 rpm in 5 mL LB broth. The bacterial culture was then plated and incubated for 24 h. The colony morphology was as follows. Figure 4 As shown, a single colony was picked and cultured overnight in 5 mL LB broth. The bacterial culture was verified by PCR (50 mL system). The PCR product with a band was sent for sequencing. The sequencing result was correct. The bacteria were preserved and streaked.

[0036] 2. Construction of recombinant Bacillus subtilis WB800N (1) Preparation of Bacillus subtilis WB800N electrocompetent cells ①Preparation: 100 mL (LB + 0.5 M sorbitol): 100 mL LB broth, 9 g sorbitol, adjust pH to 7.2, autoclave.

[0037] 600 mL electroporation medium (0.5 M trehalose, 0.5 M sorbitol, 0.5 M mannitol, 10% glycerol): 113.5 g trehalose, 54 g sorbitol, 55.5 g mannitol, 600 mL 10% glycerol, autoclaved at 115℃ for 20 min, pre-cooled at 4℃ for later use.

[0038] 100 mL RM (100 mL LB broth, 0.5 M sorbitol, 0.38 M mannitol): 100 mL LB broth, 9 g sorbitol, 7 g mannitol, autoclaved at 115°C for 20 min.

[0039] Two 50 mL centrifuge tubes were sterilized.

[0040] Add chloramphenicol at a total concentration of 3.4 μg / mL to a 100 mL LB agar plate.

[0041] ② Inoculate Bacillus subtilis WB800N in 5 mL LB broth and incubate overnight.

[0042] ③ Take 1 mL of overnight culture and inoculate it into 100 mL (LB + 0.5 M sorbitol), incubate at 37℃ and 200 rpm for 24 h (OD). 600 =2.2~2.3).

[0043] ④ Incubate the bacterial culture in an ice water bath for 30 min, then centrifuge at 4000×g for 10 min at 4℃ to collect the bacterial cells.

[0044] ⑤ Resuspend the bacterial cells in 40 mL of pre-cooled electroporation medium, centrifuge at 4000 g for 10 min at 4℃, discard the supernatant, resuspend the bacterial cells in 20 mL of pre-cooled electroporation medium, centrifuge at 4000 g for 10 min at 4℃, discard the supernatant, resuspend the bacterial cells in 10 mL of pre-cooled electroporation medium, centrifuge at 4000 g for 10 min at 4℃, discard the supernatant, resuspend the bacterial cells in 5 mL of pre-cooled electroporation medium, centrifuge at 4000 g for 10 min at 4℃, discard the supernatant. The bacterial cells are Bacillus subtilis WB800N electroporation competent cells.

[0045] (2) The preserved vector pHT43 and the recombinant plasmid pHT43-PFH constructed in Example 1 were subjected to plasmid extraction to obtain the vector plasmids pHT43 and pHT43-PFH.

[0046] (3) OD of Bacillus subtilis WB800N competent cells 600 Effect of electroporation on the conversion rate of recombinant plasmids OD of Bacillus subtilis WB800N competent cells 600 The effect on the conversion rate of electroporation recombinant plasmids is as follows: Figure 5 As shown, other electric shock parameters: electric shock voltage 2.1 kV, 2 mm, 1 electric shock, electric shock result: time constant = (5~5.8 ms), in the prepared Bacillus subtilis WB800N competent cells OD 600 At a value of 2.0, it exhibits good electrostatic conversion efficiency.

[0047] (4) Recombinant plasmid pHT43-PFH and vector plasmid pHT43 were electroporated into Bacillus subtilis WB800N Add 10 μL of recombinant plasmid pHT43-PFH and vector plasmid pHT43 (plasmid concentration > 200 ng / μL) to 100 μL of Bacillus subtilis WB800N competent cells, respectively, and incubate on ice for 10 min. Then add the cells to a pre-chilled electroporation cuvette (2 mm) and electroporate once. Electroporation parameters: 2.4 KV, 2 mm, 1 electroporation (electroporation result: time constant = (5~5.8 ms(1)). If the time constant < 5.0, it is necessary to increase the number of rinses of the electroporation medium or increase the dilution factor of the competent cells to obtain a higher conversion rate). After electroporation, remove the cuvette and immediately add 1 mL of RM. Incubate at 37℃, 200 rpm, and recover for 6 h. Then plate the cells and incubate overnight at 37℃ to obtain recombinant Bacillus subtilis WB800N / pHT43 and recombinant Bacillus subtilis WB800N / pHT43-PFH.

[0048] 3. PCR identification of recombinant Bacillus subtilis WB800N / pHT43 and recombinant Bacillus subtilis WB800N / pHT43-PFH bacterial cultures (1) Recombinant Bacillus subtilis WB800N / pHT43 and recombinant Bacillus subtilis WB800N / pHT43-PFH were screened using chloramphenicol. The screening results are as follows: Figure 6 As shown, the recombinant bacteria produced several single positive clones on the plate. The colony morphology was consistent with that of Bacillus subtilis WB800N, and there was no contamination. Six single colonies were randomly picked from the plate for subsequent PCR verification to confirm whether the recombinant plasmid had been successfully transformed into Bacillus subtilis WB800N.

[0049] (2) Recombinant Bacillus subtilis strains WB800N / pHT43 and WB800N / pHT43-PFH were picked from the chloramphenicol resistance screening plate and cultured overnight at 37°C and 220 rpm in LB broth containing 3.4 μg / mL chloramphenicol. The bacterial culture was used for bacterial culture PCR verification. The PCR amplification reaction system is shown in Table 4.

[0050] Table 4 PCR amplification reaction system PCR reaction procedure: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for 35 cycles; then 72℃ extension for 10 min, and store at 4℃.

[0051] Take 10 μL of the amplification product and perform 2% agarose gel electrophoresis. Take pictures with a gel imaging system. The results are as follows. Figure 7 As shown, the pHT43 fragment is 433 bp in size, and the target fragment is 670 bp in size. Nucleic acid electrophoresis results confirmed the transformation was consistent with the positive control, indicating that the vector plasmid pHT43 and the recombinant plasmid pHT43-PFH were successfully transformed into *Bacillus subtilis* WB800N. The gene sequence of the positive recombinant *Bacillus subtilis* strain was determined. Finally, the correctly identified recombinant *Bacillus subtilis* strains were numbered and stored at -80℃, namely recombinant *Bacillus subtilis* WB800N / pHT43 and recombinant *Bacillus subtilis* WB800N / pHT43-PFH.

[0052] 4. Preparation of antibacterial recombinant bacteriocin PFH (antibacterial recombinant protein PFH) (1) Take the recombinant Bacillus subtilis WB800N / pHT43-PFH glycerol bacteria stored at -80℃ and inoculate it into 5 mL LB broth at a ratio of 1:100. Activate the bacterial solution overnight at 37℃ and 220 rpm. Take the activated bacterial solution and streak it on a 3.4 μg / mL chloramphenicol-resistant LB agar plate according to the three-zone streak method until a single colony grows. The colony morphology is 1~2 mm in diameter, round, with irregular edges, opaque, grayish-white on the front, flat, bright, smooth, moist, and easy to pick up.

[0053] (2) Pick a single colony and place it in 5 mL of LB broth. Incubate the bacterial solution overnight at 37°C and 220 rpm. Transfer the activated bacterial solution to 500 mL of LB broth at a volume ratio of 1:100 and incubate at 37°C and 220 rpm until the bacterial solution reaches OD. 600 When the pH reaches approximately 0.8-1.0, add 0.3 mmol / L IPTG, incubate at 30℃ and 200 rpm for 48 h, and collect the fermentation supernatant at 8000 rpm for 5 min. The fermentation supernatant is the antibacterial recombinant bacteriocinolone PFH.

[0054] (3) Add 70% ammonium sulfate to the fermentation supernatant and dissolve it completely (stir at room temperature for 2 h). Precipitate the protein products in the fermentation supernatant overnight at 4℃ to obtain crude protein extract and protein precipitate. Dissolve the protein precipitate in 3 mL of 1×PBS for later use.

[0055] (4) The fermentation supernatant was filtered through a 0.22 μm filter membrane and then used for nickel column purification of the protein to obtain the purified target protein PFH, namely the purified antibacterial recombinant bacteriocin PFH.

[0056] (5) Add the purified and unpurified antibacterial recombinant bacteriocin PFH to 2× protein loading buffer (containing DTT) (Solarbio, P1018) to prepare protein samples. Place the electrophoresis tank in 4℃ or an ice water bath. Add the anode buffer (Solarbio, T1225) to the outer tank and the cathode buffer (Solarbio, T1215) to the inner tank. Perform pre-electrophoresis at 30 V for 10 min. Add the protein samples to the wells and perform electrophoresis at 30 V for 1 hour. Stop electrophoresis at 100 V until the bromophenol blue reaches the bottom of the gel. Perform subsequent Coomassie brilliant blue staining or electroporation. Analyze the size of the target protein using PAGE and Western blot.

[0057] The results are as follows Figure 8As shown, compared to the fermentation supernatant (FS), the flow through (FT) contained no target protein band, indicating that the recombinant antibacterial bacteriocin PFH was completely attached to the column. When eluted with 50 mM imidazole, a target protein band was observed at a low concentration of 8 kDa, indicating that the recombinant antibacterial bacteriocin PFH was successfully expressed. When eluted with 100 mM imidazole, no target protein was eluted, indicating that 50 mM imidazole could completely elute the recombinant antibacterial bacteriocin PFH.

[0058] 5. Bradford method for determining the protein concentration of fermentation products Recombinant Bacillus subtilis WB800N / pHT43-PFH was fermented at 30℃ for 24 h, 48 h, 72 h, 96 h, and 120 h to obtain crude fermentation extracts (antibacterial recombinant bacteriocin PFH) under each condition. The crude fermentation extract of Bacillus subtilis WB800N was used as a negative control. The protein concentration of the crude fermentation extracts under each fermentation condition was determined using the Bradford method. The results are shown in Table 5.

[0059] Table 5. Standard curve for determining protein concentration in fermentation products using the Bradford method: y = 0.4862x + 0.4981; R 2 =0.9856 The results are shown in Table 5 and Figure 9 As shown, different induction times have a significant impact on the yield of recombinant antibacterial bacteriocin PFH. Figure 9 The Oxford Cup antimicrobial susceptibility test results showed that recombinant Bacillus subtilis WB800N / pHT43-PFH exhibited the best antibacterial effect against Staphylococcus aureus ATCC-29213 after induction at 30℃ for 48 h and 72 h, with an inhibition zone diameter of 15 mm and a protein concentration (Table 5) of over 10 μg / mL. After induction at 30℃ for 96 h and 120 h, the antibacterial effect of PFH against Staphylococcus aureus ATCC-29213 was weak, with an inhibition zone diameter of 11–13 mm and a protein concentration (Table 5) of 7.59–7.85 μg / mL. Therefore, the protein concentration range of the antimicrobial recombinant bacteriocin PFH in recombinant Bacillus subtilis WB800N / pHT43-PFH is 8–10 μg / mL and above.

[0060] Example 3: Method for determining antibacterial activity (1) Resuscitation indicator bacteria Escherichia coli ATCC-25922 and Staphylococcus aureus ATCC-29213 were streaked and incubated overnight at 37°C. Typical single colonies were picked and inoculated into test tubes containing 5 mL of LB liquid medium and incubated at 37°C and 220 rpm for 3-4 h until OD. 600 =0.4~0.6 (1OD) 600 =3~5×10 7 (Cells / mL).

[0061] (2) Pour about 15 mL of lower agar into a 90 mm petri dish and wait for it to solidify. Then place the Oxford cups according to the number of samples.

[0062] (3) Take about 20 mL of LB agar. When the temperature of LB agar drops below 50°C, inoculate the pre-activated indicator bacteria at a volume ratio of 1:1000 between LB agar and indicator bacteria. Shake well and pour the top layer of agar.

[0063] (4) After the upper agar solidifies, remove the Oxford cup, mark it, and add 100 μL of the crude fermentation extract of recombinant Bacillus subtilis WB800N / pHT43-PFH (antibacterial recombinant bacteriocin PFH) to each well according to the marked well. Place it at 4℃ for 4 h. After the liquid is absorbed, place it in a 37℃ incubator and incubate overnight. The antibacterial effect is judged according to the size of the inhibition zone radius.

[0064] The results are as follows Figure 21 As shown, the antibacterial effects of the crude fermentation extracts of Bacillus subtilis WB800N, WB800N / pHT43, and recombinant Bacillus subtilis WB800N / pHT43-PFH were determined using the Oxford cup antimicrobial susceptibility testing method. The results showed that the crude fermentation extracts of the negative control group WB800N and WB800N / pHT43 had no inhibitory effect on Escherichia coli ATCC-25922 and Staphylococcus aureus ATCC-29213. The crude fermentation extract of recombinant Bacillus subtilis WB800N / pHT43-PFH (antimicrobial recombinant bacteriocin PFH) had no inhibitory effect on Escherichia coli ATCC-25922, and the inhibition zone diameter against Staphylococcus aureus ATCC-29213 was 22 mm.

[0065] Example 4: Optimization of expression conditions for recombinant Bacillus subtilis WB800N At the optimal induction time, univariate analysis was conducted on the expression levels of antibacterial recombinant bacteriocin PFH under different IPTG concentrations (0.1, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, 1.5 mmol / L), different induction times (0, 1.5, 2, 3, 4, 5, 6, 7, 9 h), and different pH culture media (4, 5, 6, 6.6, 7, 8, 9) to determine the optimal induction expression conditions.

[0066] 1. Determination of the growth curve of recombinant Bacillus subtilis WB800N (1) The constructed recombinant Bacillus subtilis WB800N / pHT43 and recombinant Bacillus subtilis WB800N / pHT43-PFH strains were inoculated into LB liquid medium containing 3.4 μg / mL chloramphenicol and cultured overnight at 37°C and 220 rpm.

[0067] (2) Inoculate the culture medium into 100 mL LB liquid medium at a ratio of 1:100 (V / V) for expansion culture. Start timing at this time and take 1 mL of bacterial solution at 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8 and 9 h for OD analysis. 600 Values ​​were measured, and growth curves of recombinant Bacillus subtilis WB800N / pHT43 and recombinant Bacillus subtilis WB800N / pHT43-PFH were plotted to determine their growth patterns.

[0068] Growth curves as follows Figure 10 As shown, in the first 9 hours, the growth of both recombinant Bacillus subtilis WB800N / pHT43 and recombinant Bacillus subtilis WB800N / pHT43-PFH showed a significant upward trend. The OD values ​​of both recombinant Bacillus subtilis WB800N / pHT43 and recombinant Bacillus subtilis WB800N / pHT43-PFH were around 5-6 hours. 600 When the value reaches 1.0 to 1.2, this is the optimal time for induction.

[0069] 2. Determination of IPTG-induced concentration Overnight-cultured recombinant Bacillus subtilis WB800N / pHT43 and recombinant Bacillus subtilis WB800N / pHT43-PFH were inoculated into 100 mL LB liquid medium at a ratio of 1:100 (V / V). 600 IPTG at concentrations of 0.1, 0.3, 0.5, 1.0, and 2.0 mmol / L was added at a concentration of 1.0, and the fermentation supernatant was collected after 60 h. The antibacterial activity of the fermentation supernatant against Staphylococcus aureus ATCC-29213 was determined using the same method as in Example 3. The optimal induction time for the antibacterial recombinant bacteriocin PFH was analyzed by examining the inhibition zone of the fermentation supernatant.

[0070] like Figure 11 As shown, when the IPTG concentration is 0.3 mmol / L, the inhibition zone of the antibacterial recombinant bacteriocin PFH is the largest, around 12 mm. Under other conditions, the inhibition zone is smaller or non-existent. Therefore, 0.3 mmol / L is the optimal induction concentration of IPTG.

[0071] 3. Determination of induction timing Recombinant Bacillus subtilis WB800N / pHT43-PFH cultured overnight was inoculated into 100 mL LB liquid medium at a ratio of 1:100 (V / V). The OD values ​​of the recombinant Bacillus subtilis WB800N / pHT43-PFH were measured at 4, 5, 6, 7, 8, and 9 hours of culture. 600 OD 600 The values ​​were 0.235, 0.474, 0.772, 0.906, 1.231, and 1.572, respectively. At each time point, 0.3 mmol / L IPTG was added, and after 60 h of induction, the protein was collected. The antibacterial activity of the recombinant bacteriocin PFH against Staphylococcus aureus ATCC-29213, Escherichia coli ATCC-25922, Bacillus polymyxa bxy, Bacillus subtilis 63501, Streptococcus suis 988S, and Enterococcus faecalis JH2-2 was determined using the same method as in Example 3. The results are as follows Figure 12 As shown, in OD 600 When the concentration is 1.0–1.2, the antibacterial recombinant bacteriocin PFH is effective against most Gram-positive bacteria (G6). ＋ It exhibits good antibacterial activity against Gram-negative bacteria (G bacteria), with an inhibition zone of approximately 17-24 mm. － It has weak antibacterial activity, with an inhibition zone of about 10-12 mm.

[0072] 4. Determination of induction time Overnight-cultured recombinant Bacillus subtilis WB800N / pHT43-PFH was inoculated into 100 mL LB liquid medium at a ratio of 1:100 (V / V). When the bacterial cells reached the logarithmic growth phase, i.e., OD... 600 After approximately 1.0, add IPTG inducer to a final concentration of 0.3 mmol / L, start timing, and take 20 mL of bacterial solution at 12, 24, 48, 72, 96, and 120 h to determine the antibacterial activity against Staphylococcus aureus. The determination method is the same as in Example 3, and the optimal induction time of the expression strain is analyzed.

[0073] The effects of different induction times on the growth of recombinant Bacillus subtilis WB800N / pHT43-PFH and the secretion of its antibacterial active substances are as follows: Figure 13 As shown, recombinant Bacillus subtilis WB800N / pHT43-PFH reached the growth plateau phase after 24 h of induction and began to secrete the expression product. At 48 h, the antibacterial activity of the expression product, recombinant bacteriocin PFH, against Staphylococcus aureus reached its maximum. Subsequent increases in induction time did not significantly change the antibacterial activity of the expression product, recombinant bacteriocin PFH, against Staphylococcus aureus. Therefore, the optimal induction time for recombinant Bacillus subtilis WB800N / pHT43-PFH is 48 h.

[0074] 5. Effects of different pH culture media on the expression level of recombinant bacteriocin PFH. Overnight-cultured recombinant Bacillus subtilis WB800N / pHT43-PFH was inoculated into 100 mL LB broth at pH 4, 5, 6, 6.6, 7, 8, and 9 at a ratio of 1:100 (V / V). When the bacterial cells reached the logarithmic growth phase (OD), the inoculum was cultured. 600 After approximately 1.0, IPTG inducer with a final concentration of 0.3 mmol / L was added, and timing was started. The fermentation supernatant was collected after 60 h, and the antibacterial activity against Staphylococcus aureus was determined. The determination method was the same as in Example 3. The optimal culture pH for the expression strain was analyzed.

[0075] The effects of different pH culture media on the growth of recombinant Bacillus subtilis WB800N / pHT43-PFH and the secretion of its antibacterial active substances are as follows: Figure 14 As shown, compared with the original culture medium at pH 6.6, adjusting the pH of the culture medium to 6 increased the inhibition zone of Staphylococcus aureus ATCC29213 by 2 mm. However, both excessively acidic and excessively alkaline culture conditions are not conducive to bacterial growth and the expression of exogenous proteins.

[0076] 6. Effects of adding different proportions of lactose to the basal culture medium on the expression level of recombinant bacteriocin PFH. Overnight-cultured recombinant Bacillus subtilis WB800N / pHT43-PFH was inoculated at a ratio of 1:100 (V / V) into 100 mL LB broth containing 0%, 1%, 2%, 3%, 4%, 5%, and 6% lactose, respectively. The inoculum was cultured until it reached the logarithmic growth phase (OD). 600 After reaching approximately 1.0, IPTG inducer with a final concentration of 0.3 mmol / L was added to LB medium containing 0% lactose, and timing was started. Fermentation supernatants under different culture conditions were collected after 60 h to determine the antibacterial activity against Staphylococcus aureus. The determination method was the same as in Example 3. The lactose content in the optimal culture medium suitable for the expression strain was analyzed.

[0077] The effects of adding different proportions of lactose to the basal culture medium on the growth of recombinant Bacillus subtilis WB800N / pHT43-PFH and the secretion of its antibacterial active substances are as follows: Figure 15 As shown, compared with the induction condition of 0.3 mmol / L IPTG, the basal medium containing 4%-5% lactose is more advantageous for the growth of recombinant strain WB800N / pHT43-PFH and the secretion of its antibacterial active substances. Too high or too low lactose content has a relatively poor effect on the secretion of antibacterial active substances by recombinant strain WB800N / pHT43-PFH.

[0078] Example 5: Stability Study of Polymyxin Bacillus-Derived Recombinant Bacteriocin PFH In the stability test of the recombinant Bacillus subtilis WB800N / pHT43-PFH expression product (antibacterial recombinant bacteriocin PFH), the stability of the antibacterial recombinant bacteriocin PFH before and after heat treatment, protease treatment, pH treatment, surfactant treatment, and treatment with metal salt ions at physiological concentrations was detected by antibacterial activity assay. The indicator bacteria selected were the Gram-negative bacterium *Escherichia coli* ATCC-25922 and the Gram-positive bacterium *Staphylococcus aureus* ATCC029213, and the antibacterial activity assay method was the same as in Example 3.

[0079] 1. The effect of temperature on the stability of recombinant bacteriocin PFH Six 2 mL sterile EP tubes were used, and 500 μL of recombinant antibacterial bacteriocin PFH (pH 7.25) was added to each tube. The tubes were treated at 20, 40, 60, 80, 100, and 120 °C for 2 h each. After cooling, if any liquid evaporated from the centrifuge tubes, sterile water was added to bring the total volume to 500 μL. 20 °C served as a control (room temperature). The experiment was repeated three times. The antibacterial activity of the treated recombinant antibacterial bacteriocin PFH against Staphylococcus aureus was determined.

[0080] Results of the thermal stability of the antibacterial recombinant bacteriocin PFH are as follows: Figure 16 As shown, the antibacterial activity of the recombinant Bacillus subtilis WB800N / pHT43-PFH expression product, recombinant antibacterial bacteriocin PFH, changed significantly after treatment at different temperatures. After heat treatment at 20, 40, 60, 80, 100, and 120℃ for 2 h, the antibacterial effect of recombinant antibacterial bacteriocin PFH against Gram-positive Staphylococcus aureus decreased progressively with increasing temperature compared to the control (20℃), indicating poor thermal stability of recombinant antibacterial bacteriocin PFH. The antibacterial activity of recombinant antibacterial bacteriocin PFH began to decrease at 60℃, and was completely lost above 100℃. These results indicate that recombinant antibacterial bacteriocin PFH should not be dried at high temperatures in subsequent production applications and requires room-temperature drying.

[0081] 2. Effects of different pH conditions on the stability of recombinant Bacillus subtilis WB800N / pHT43-PFH expression product Crude extracts of recombinant recombinant bacteriocin PFH, obtained by ammonium sulfate precipitation of recombinant Bacillus subtilis WB800N / pHT43-PFH fermentation broth, were subjected to pH adjustments to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13, respectively. After treatment at 37℃ for 2 h in a water bath, the pH was adjusted back to 7. Untreated crude extracts of recombinant recombinant bacteriocin PFH served as a blank control. The experiment was repeated three times. The antibacterial activity of the treated recombinant bacteriocin PFH against Staphylococcus aureus was determined.

[0082] The pH stability of the recombinant Bacillus subtilis WB800N / pHT43-PFH expression product is as follows: Figure 17 As shown, the results indicate that the antibacterial activity of the recombinant bacteriocin PFH did not change significantly after treatment under different pH conditions at 37℃. Figure 17 As shown in Figure A, treatment with antibacterial substances at pH 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, and 13 for 2 hours did not result in a significant difference in antibacterial efficacy against Staphylococcus aureus compared to the control (pH 7.0). Figure 17 As can be seen from B, pH changes have little effect on the stability of recombinant antibacterial bacteriocin PFH. That is, at 37℃, the pH stability of recombinant antibacterial bacteriocin PFH is very good, and it exhibits high stability in the pH range of 2 to 12.

[0083] 3. Effects of proteolytic enzymes on the stability of recombinant Bacillus subtilis WB800N / pHT43-PFH expression products (1) Prepare pepsin, trypsin, papain, proteinase K, catalase, creatine kinase and α-chymotrypsin (1000 U / mg), each enzyme solution 5 mg / mL. Take 0.1 mL of enzyme solution and place it in a centrifuge tube. Add 0.4 mL of antibacterial recombinant bacteriocin PFH that has been adjusted to the optimal pH value of each enzyme. Shake well and incubate in a 37°C water bath for 2 h. Adjust the pH value to 4 with NaOH or HCl. Test the antibacterial activity of the treated antibacterial recombinant bacteriocin PFH against Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 29213). Use the treatment with added enzyme-free buffer as a blank control. Repeat the test three times.

[0084] (2) Prepare pepsin, trypsin, papain, proteinase K, catalase, creatine kinase and α-chymotrypsin (1000 U / mg), each enzyme solution 5 mg / mL. Take 0.2 mL of enzyme solution and place it in a centrifuge tube. Add 0.3 mL of antibacterial recombinant bacteriocin PFH that has been adjusted to the optimal pH value of each enzyme. Shake well and incubate in a 37°C water bath for 2 h. Adjust the pH value to 4 with NaOH or HCl. Test the antibacterial activity of the treated antibacterial recombinant bacteriocin PFH against Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 29213). Use the treatment with added enzyme-free buffer as a blank control. Repeat the test three times.

[0085] The results are as follows Figure 18 As shown, Figure 18 Results showed that after treatment with 1 mg / mL chymotrypsin (>1000 U / mL), the inhibition zone of the recombinant antibacterial bacteriocin PFH was reduced by 2 mm compared to the blank control group. Treatment with other proteases did not significantly alter the antibacterial activity of the recombinant antibacterial bacteriocin PFH. Figure 18 As shown in Figure B, after treatment with 2 mg / mL trypsin (>1000 U / mL), the inhibition zone of the recombinant antibacterial bacteriocin PFH decreased by 3-4 mm compared to the blank control group. Treatment with other proteases did not significantly alter the antibacterial activity of the recombinant antibacterial bacteriocin PFH. These results indicate that antibacterial substances are unstable against chymotrypsin and trypsin, and can be degraded under certain conditions, thus losing their antibacterial activity. Furthermore, the lack of significant changes in the antibacterial activity of the recombinant antibacterial bacteriocin PFH after treatment with other proteases suggests that the 37℃ water bath condition had no effect on the stability of the recombinant antibacterial bacteriocin PFH.

[0086] 4. Effects of different UV irradiation times on the stability of recombinant Bacillus subtilis WB800N / pHT43-PFH expression products Take seven 2 mL sterile EP tubes and add 500 μL of recombinant antibacterial bacteriocin (pH 7.25) to each tube. Treat the tubes with ultraviolet (UV) irradiation for 0.5, 1, 2, 3, 4, and 5 h at room temperature. Repeat the experiment three times. Determine the antibacterial activity of the treated recombinant antibacterial bacteriocin PFH against Staphylococcus aureus.

[0087] The effect of UV on the stability of recombinant Bacillus subtilis WB800N / pHT43-PFH expression product, such as Figure 19 As shown, the antibacterial recombinant bacteriocin PFH, after treatment under ultraviolet (UV) irradiation for 0.5, 1, 2, 3, 4, and 5 h, had no significant effect on the antibacterial activity against Staphylococcus aureus compared with the blank control (antibacterial recombinant bacteriocin PFH without UV irradiation treatment).

[0088] 5. Effects of metal salt ions at physiological concentrations on the stability of recombinant Bacillus subtilis WB800N / pHT43-PFH expression products Solutions of CaCl2, NaCl, KCl, NH4Cl, MgCl2, ZnCl2, and FeCl3 with final concentrations of 2.5 mmol / L, 150 mmol / L, 4.5 mmol / L, 6 mmol / L, MgCl2, ZnCl2, and FeCl3 were mixed with antibacterial recombinant bacteriocin PFH dilutions with the same final concentration gradient as the MIC. The antibacterial activity of the recombinant bacteriocin PFH against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 in physiological saline was determined. The crude extract of the recombinant bacteriocin PFH without salt ion treatment was used as a control. The experiment was repeated three times.

[0089] The effect of metal salt ions at physiological concentrations on the stability of recombinant Bacillus subtilis WB800N / pHT43-PFH expression products as follows: Figure 20 As shown, solutions with final concentrations of 2.5 mmol / L CaCl2, 150 mmol / L NaCl, 4.5 mmol / L KCl, 1 mmol / L MgCl2, and 4 mmol / L FeCl3 had no significant effect on the activity of the recombinant bacteriocin PFH against Staphylococcus aureus.

[0090] 6. Effect of surfactants on the stability of recombinant Bacillus subtilis WB800N / pHT43-PFH expression products A crude extract of recombinant bacteriocin PFH containing 8 mM solutions of Tris, SDS, TW-80, TW-20, EDTA, methanol, and acetonitrile was prepared, dispensed into 1.5 mL sterile EP tubes, and incubated at 37°C for 2 h. The antibacterial activity of the recombinant bacteriocin PFH in the reaction solution against Staphylococcus aureus was then determined. The residual antibacterial activity was calculated using a recombinant bacteriocin solution without added surfactant as a control. The experiment was repeated three times.

[0091] The effect of 8 mM surfactant on the stability of recombinant Bacillus subtilis WB800N / pHT43-PFH expression product is as follows: Figure 21 As shown, the addition of TW-80, SDS, and acetonitrile reduces the inhibitory effect of recombinant bacteriocin PFH on Staphylococcus aureus. Compared with the negative control, solvents such as Tris, TW-20, EDTA, and methanol have little or no effect on the activity of recombinant bacteriocin PFH.

[0092] Example 6: Determination of the antibacterial effect of recombinant bacteriocin PFH The antibacterial activity of recombinant bacteriocin PFH against Gram-positive and Gram-negative bacteria was determined using the same method as in Example 3. Simultaneously, the antibacterial activities of bacitracin, polymyxin B, and vancomycin were measured as controls. Gram-positive bacteria included Staphylococcus aureus ATCC-29213, Micrococcus luteus, Enterococcus faecalis JH2-2, Streptococcus suis ATCC 43765, Streptococcus suis 988S, Bacillus subtilis WB800N, Bacillus subtilis 63501, and Bacillus polymyxin bxy. Gram-negative bacteria included Escherichia coli ATCC-25922, Salmonella SH134, and Pseudomonas aeruginosa.

[0093] The antibacterial recombinant bacteriocin PFH and different concentrations of peptide drugs, including commercially available bacitracin (Solarbio), commercially available polymyxin B (Solarbio), and commercially available vancomycin (Solarbio), were compared with several Gram-positive bacteria. ＋ and G － The results of the comparative study on the antibacterial effects of representative strains are shown in Tables 6, 7, and 8. The antibacterial recombinant bacteriocin PFH against G... ＋ The antibacterial activity of recombinant bacteriocins (such as Staphylococcus aureus ATCC-29213, Enterococcus faecalis JH2-2, Streptococcus suis ATCC 43765, Streptococcus suis 988S, Bacillus subtilis WB800N, Bacillus subtilis 63501, and Bacillus polymyxa bxy) was generally significantly superior to that of polymyxin B and bacitracin. The antibacterial recombinant bacteriocin PFH was effective against most Gram-positive bacteria. ＋ The antibacterial activity of the bacteria (Staphylococcus aureus ATCC-29213, Streptococcus suis 988S, Bacillus subtilis 63501, and Enterococcus faecalis JH2-2) was superior to that of vancomycin at 512 μg / mL, 256 μg / mL, 64 μg / mL, and 128 μg / mL, respectively.

[0094] Table 6. Antimicrobial recombinant bacteriocin PFH and different concentrations of polymyxin B against G. ＋ and G － Comparison of antibacterial effects of representative strains Table 7. Effects of different concentrations of recombinant bacteriocin PFH on G... ＋ and G － Comparison of antibacterial effects of representative strains Table 8. Effects of recombinant bacteriocin PFH and different concentrations of vancomycin on Gram-positive bacteria. ＋ and G － Comparison of antibacterial effects of representative strains Example 7 Preparation of antibacterial agent The antibacterial agent in this embodiment contains recombinant antibacterial bacteriocin PFH, and the concentration of recombinant antibacterial bacteriocin PFH in the antibacterial agent is 8~10μg / mL.

[0095] The antibacterial recombinant bacteriocin PFH was prepared from the fermentation supernatant of recombinant Bacillus subtilis WB800N / pHT43-PFH, using the same method as in Example 2.

[0096] Comparative Example 1: Preparation of Fermentation Broth 1 Fermentation broth 1 in this embodiment is the fermentation supernatant of recombinant Bacillus subtilis WB800N / pHT43, and the preparation method is the same as in Example 2.

[0097] Comparative Example 2: Preparation of Fermentation Broth 2 Fermentation broth 2 in this embodiment is the fermentation supernatant of Bacillus subtilis WB800N, and the preparation method is the same as in embodiment 2.

[0098] Example 8: Determination of antibacterial activity and antibacterial spectrum of antibacterial agent The test strains were the standard quality control strains: Staphylococcus aureus ATCC-29213, Escherichia coli ATCC-25922, Micrococcus luteus, Enterococcus faecalis JH2-2, and Streptococcus suis ATCC 43765.

[0099] 1. Determination of antibacterial activity of antibacterial agents The antimicrobial activity of the antimicrobial agent, fermentation broth 1, and fermentation broth 2 was determined using the Oxford cup method for antimicrobial susceptibility testing. *Escherichia coli* ATCC-25922 and *Staphylococcus aureus* ATCC-29213 were used as test strains, and the procedure was performed as follows: (1) Resuscitate frozen Escherichia coli ATCC-25922 and Staphylococcus aureus ATCC-29213, streak and incubate overnight at 37°C, pick typical single colonies and inoculate into test tubes containing 5 mL LB liquid medium, and incubate at 37°C and 220 rpm for 3-4 h until OD. 600 =0.4~0.6 (1OD) 600 =3~5×10 7 (Cells / mL).

[0100] (2) Pour about 15 mL of lower agar into a 90 mm petri dish and wait for it to solidify. Then place the Oxford cups according to the number of samples.

[0101] (3) Take about 20 mL of agar. When the agar temperature drops below 50℃, inoculate the pre-activated quality control bacteria at a volume ratio of 1:1000 between agar and quality control bacteria. Shake well and pour the top layer of agar.

[0102] (4) After the upper agar solidifies, remove the Oxford cup, mark it, add 100 μL of antibacterial agent to each well according to the marked well, and add fermentation broth 1 and fermentation broth 2 as controls. Place at 4℃ for 4 h. After the liquid is absorbed, place in a 37℃ incubator and incubate overnight. The antibacterial effect is judged according to the size of the inhibition zone radius.

[0103] Oxford cup antibacterial test results as follows Figure 22 As shown, fermentation broth 1 and fermentation broth 2 had no inhibitory effect on either of the two quality control strains. However, the antibacterial agent had a better inhibitory effect on the quality control strain Staphylococcus aureus ATCC29213, with an inhibition zone of 5-6 mm.

[0104] 2. Determination of the antibacterial spectrum of antibacterial agents Antimicrobial activity was determined using the Oxford cup method for drug susceptibility testing. The standard control strains and clinical isolates included Staphylococcus aureus ATCC-29213, Micrococcus luteus, Enterococcus faecalis JH2-2, and Streptococcus suis ATCC 43765. The procedure was performed as follows: (1) Resuscitate the frozen strains of the above test strains, streak them at 37°C and incubate overnight, pick typical single colonies, inoculate them into liquid culture medium containing 5 mL, and incubate at 37°C and 220 rpm until the logarithmic growth phase.

[0105] (2) Pour about 15 mL of lower agar into a 90 mm petri dish and wait for it to solidify. Then place the Oxford cups according to the number of samples.

[0106] (3) Take about 20 mL of agar. When the agar temperature drops below 50℃, inoculate the pre-activated test strain at a volume ratio of 1:1000 between agar and test strain. Shake well and pour the top layer of agar.

[0107] (4) After the upper agar solidifies, remove the Oxford cup, mark it, add 100 μL of antibacterial agent to each well according to the marking, and add different concentrations of bacitracin (64, 128, 256 and 512 μg / mL respectively) as controls. Place at 4℃ for 4h, and after the liquid is absorbed, place in a 37℃ incubator for overnight incubation. The antibacterial effect is judged according to the size of the inhibition zone radius.

[0108] Results of antibacterial agent antibacterial spectrum study: Figure 23 As shown, the antibacterial agent had no inhibitory activity against Micrococcus luteus, but showed good inhibitory activity against Streptococcus suis ATCC 43765. However, its inhibitory activity was worse than that of 64 μg / mL bacitracin. The antibacterial agent was more effective than 512 μg / mL bacitracin against Staphylococcus aureus ATCC-29213 and Enterococcus faecalis JH2-2.

[0109] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A bacteriocin PA, characterized in that: The amino acid sequence of the bacteriocin PA is shown in SEQ ID NO:

2.

2. A nucleotide sequence encoding bacteriocin PA is shown in SEQ ID NO:

1.

3. A method for constructing recombinant Bacillus subtilis WB800N, characterized in that: Includes the following steps: (1) The nucleotide sequence of bacteriocin PA was obtained by the online bacteriocin prediction website BAGEL4. The 5′ end of the nucleotide sequence was linked to the ribosome binding site RBS, and the 3′ end was linked to the 3×flag and 6×His tags respectively. After codon optimization, the antibacterial recombinant bacteriocin gene PFH was obtained. (2) The antibacterial recombinant bacteriocin gene PFH was ligated into the pHT43 vector. BamH I and Sma Between the restriction enzyme sites, the plasmid was transferred into E. coli, and the plasmid was extracted to obtain the recombinant plasmid pHT43-PFH; (3) The pHT43-PFH plasmid was electroporated into Bacillus subtilis WB800N, and recombinant Bacillus subtilis WB800N was obtained after screening.

4. The construction method according to claim 3, characterized in that: In step (1), the nucleotide sequence of the antibacterial recombinant bacteriocin gene PFH is shown in SEQ ID NO: 3; the amino acid sequence of the antibacterial recombinant bacteriocin gene PFH is shown in SEQ ID NO:

4.

5. A method for preparing an antibacterial recombinant bacteriocin PFH, characterized in that: Includes the following steps: The recombinant Bacillus subtilis WB800N plate constructed by the method described in claim 3 was streaked and cultured. Single colonies were picked and placed in LB broth for activation. The activated bacterial solution was transferred into LB broth and cultured with shaking to expand the culture. Isopropyl-β-D-thiogalactoside was added to induce expression. The fermentation supernatant was collected by centrifugation. The fermentation supernatant was the antibacterial recombinant bacteriocinol PFH.

6. The preparation method according to claim 5, characterized in that: The conditions for culture and activation were 37℃, 220 rpm, and 8–12 h. The activated bacterial solution was transferred into LB broth at a volume ratio of 1:100, and cultured with shaking at 37°C and 220 rpm until the bacterial solution reached its OD value. 600 To 0.8~1.2; Isopropyl-β-D-thiogalactoside was added to a final concentration of 0.1–2 mmol / L, and the induction conditions were 28–32 °C, 200 rpm, and 48–72 h.

7. The application of the antibacterial recombinant bacteriocin PFH prepared by the preparation method as described in claim 5 in inhibiting bacterial activity, characterized in that: The bacteria are any one of Staphylococcus aureus, Micrococcus luteus, Enterococcus faecalis, Streptococcus suis, Bacillus subtilis, and Bacillus polymyxa.

8. The application of the antibacterial recombinant bacteriocin PFH prepared by the preparation method as described in claim 5 in the preparation of antibacterial agents.

9. An antibacterial agent, characterized in that: The antibacterial agent comprises the antibacterial recombinant bacteriocin PFH prepared by the preparation method as described in claim 5, wherein the concentration of the antibacterial recombinant bacteriocin PFH in the antibacterial agent is 8~10 μg / mL.

10. The use of the antibacterial agent as described in claim 9 in inhibiting bacterial activity, characterized in that: The bacteria are any one of Staphylococcus aureus, Enterococcus faecium, and Streptococcus suis.

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