Streptococcus salivarius antibacterial peptide, and preparation method and application thereof

Abstract

The application discloses a Streptococcus salivarius antibacterial peptide and a preparation method and application thereof. The Streptococcus salivarius antibacterial peptide is a tandem peptide formed by two Salivaricin B small peptides in series, or Salivaricin B and Salivaricin A2 small peptides in series. The Streptococcus salivarius antibacterial peptide is formed by two Salivaricin B small peptides in series, or Salivaricin B and Salivaricin A2 in series, and the antibacterial effect of the tandem peptide is obviously superior to that of the single small peptide compared with the single Salivaricin B small peptide or Salivaricin A2 small peptide. The Streptococcus salivarius antibacterial peptide provides a more effective scheme and approach for the prevention and treatment of oral diseases caused by pathogenic bacteria.

Description

Technical Field

[0001] This application relates to the field of oral bacteria technology, and in particular to an antimicrobial peptide of Streptococcus salivarius, its preparation method and application. Background Technology

[0002] Microorganisms colonizing the human body, along with human cells, together constitute functional organs and maintain human physiological activities. The development of next-generation sequencing technology has propelled research into the human microbiome. The warm, moist environment of the oral cavity provides conditions for microbial growth, and the oral cavity is also a way for microorganisms to enter the body. Therefore, oral health plays a vital role in human health. However, current human oral microbiome databases only contain 775 bacterial species. By isolating bacteria from different oral samples and different oral sites, this study aims to confirm the composition of oral microorganisms and the differences between the oral flora of patients and healthy individuals, providing guidance for the clinical treatment of oral diseases.

[0003] In the treatment of oral diseases, the overuse of antibiotics has led to the increasingly frequent emergence of drug-resistant bacteria, making the search for new antibiotics increasingly urgent. The discovery of antimicrobial peptides offers a new solution for drug-resistant bacteria. Antimicrobial peptides, also known as host defense peptides, are short, naturally occurring immune peptides with high activity, good thermal stability, tolerance across a wide pH range, and a broad spectrum of antimicrobial activity that is less likely to induce drug resistance.

[0004] However, there is relatively little research on antimicrobial peptides at present, and the role of antimicrobial peptides in oral flora remains a key research focus and challenge in this field. Summary of the Invention

[0005] The purpose of this application is to provide a novel antimicrobial peptide for saliva streptococci, its preparation method, and its application.

[0006] The following technical solution is adopted in this application:

[0007] One aspect of this application discloses a saliva streptococcal antimicrobial peptide, which is a tandem peptide composed of two small peptides of saliva varicin B, or saliva varicin B and saliva varicin A2.

[0008] Preferably, the antimicrobial peptide of *Streptococcus salivarius* is *Salivaricin B+Salivaricin B tandem peptide*, *Salivaricin B+Salivaricin A2 tandem peptide*, or *Salivaricin A2+Salivaricin B tandem peptide*.

[0009] Preferably, the two small peptides are linked by a linker arm.

[0010] Preferably, the sequence of the connecting arms is "GSGGSG".

[0011] For example, the antimicrobial peptide of *Streptococcus salivarius* is Salivaricin B-GSGGSG-Salivaricin B, Salivaricin B-GSGGSG-Salivaricin A2, or Salivaricin A2-GSGGSG-Salivaricin B. It is understood that "GSGGSG" is merely one implementation of the linker used in this application, and other amino acid sequences may also be used for linking.

[0012] Another aspect of this application discloses the use of the salivary streptococcal antimicrobial peptide in the preparation of a medicament for treating oral diseases.

[0013] Another aspect of this application discloses the use of the salivary streptococcal antimicrobial peptide of this application in the preparation of a reagent that inhibits at least one of Streptococcus griseus, Streptococcus sanguinis, and Streptococcus mutans.

[0014] Another aspect of this application discloses the use of the salivary streptococcal antimicrobial peptide of this application in the preparation of a medicament for treating diseases caused by at least one of Streptococcus griseus, Streptococcus sanguinis, and Streptococcus mutans.

[0015] Another aspect of this application discloses a drug containing the salivary streptococcal antimicrobial peptide of this application.

[0016] Preferably, the drug of this application includes the following uses:

[0017] (1) Used to treat oral diseases;

[0018] (2) Used to inhibit the growth of at least one of Streptococcus griseus, Streptococcus sanguinis, and Streptococcus mutans;

[0019] (3) Used to treat diseases caused by at least one of Streptococcus griseus, Streptococcus sanguinis, or Streptococcus mutans.

[0020] Another aspect of this application discloses a method for preparing the *Streptococcus salivarius* antimicrobial peptide, comprising: introducing the gene encoding *Salivaricin B*, or *Salivaricin B* and *Salivaricin A2*, into a plasmid according to a tandem peptide design to construct a recombinant plasmid; transforming the recombinant plasmid into competent cells to express the target protein, thereby obtaining the *Streptococcus salivarius* antimicrobial peptide. It is understood that if a linker arm is present, the gene sequence of the linker arm also needs to be constructed into the recombinant plasmid.

[0021] Preferably, the plasmid is an Escherichia coli plasmid.

[0022] Preferably, the plasmid is pGEX-6P-1.

[0023] Preferably, the competent cells are cells from an Escherichia coli expression system, a yeast expression system, or a lactic acid bacteria expression system.

[0024] Preferably, competent cells are prepared from Escherichia coli.

[0025] Preferably, the preparation method of this application further includes, after transforming the recombinant plasmid into competent cells, inducing the expression of the target protein under the action of an inducer; and lysing the cells to separate and purify the Streptococcus salivarius antimicrobial peptide.

[0026] The beneficial effects of this application are as follows:

[0027] The antimicrobial peptide of this application comprises two tandem Salivaricin B peptides, or Salivaricin B and Salivaricin A2, and exhibits significantly superior antibacterial efficacy compared to individual Salivaricin B or Salivaricin A2 peptides. This antimicrobial peptide of the application provides a more effective treatment approach for oral diseases. Attached Figure Description

[0028] Figure 1 This is an electrophoresis diagram of the recombinant plasmid and enzyme digestion identification in the embodiments of this application;

[0029] Figure 2 This is a figure showing the results of SDS-PAGE and Western blot analysis of the expression of the target protein in E. coli BL21(DE3) strain in the embodiments of this application;

[0030] Figure 3 This is a diagram showing the results of expression identification, optimization, and solubility analysis of the tandem peptides in the embodiments of this application;

[0031] Figure 4 This is a graph showing the results of SDS-PAGE analysis of the separation and purification of BA2, BB, A2A2, and A2B in the embodiments of this application.

[0032] Figure 5 and Figure 6 This is a graph showing the analysis results of the effect of BA2 antimicrobial peptide on the growth curve of Streptococcus griseus in the embodiments of this application;

[0033] Figure 7 and Figure 8 This is a graph showing the analysis results of the effect of BA2 antimicrobial peptide on the growth curve of Streptococcus sanguinis in the embodiments of this application;

[0034] Figure 9 and Figure 10 This is a graph showing the analysis results of the effect of BA2 antimicrobial peptide on the growth curve of Streptococcus mutans in the embodiments of this application;

[0035] Figure 11 This is a schematic diagram of the three-dimensional structure of the final effector peptide of Salivaricin A2 in the embodiments of this application;

[0036] Figure 12 This is a schematic diagram of the sequence modification structure of the final effector peptide of Salivaricin A2 in the embodiments of this application;

[0037] Figure 13 This is a schematic diagram of the sequence modification structure of the final effector peptide of Salivaricin B in the embodiments of this application. Detailed Implementation

[0038] This application utilizes culture techniques to separate samples from different oral cavity sites and locations, providing a basis for understanding the distribution of oral microorganisms. Secondly, it employs heterologous expression technology to achieve prokaryotic expression of antimicrobial peptides from *Streptococcus salivarius*, obtaining large quantities of purified antimicrobial peptides. These peptides are then used in vitro to act on harmful bacteria isolated from the oral cavity, investigating the effects of the antimicrobial peptides on the oral microbiota.

[0039] This application successfully constructed recombinant plasmids pGEX-6P-1-sal B, pGEX-6P-1-sal A2, pGEX-6P-1-BA2, pGEX-6P-1-A2A2, pGEX-6P-1-A2B, and pGEX-6P-1-BB, successfully transformed them into E.coli BL21(DE3) competent cells to construct expression vectors, and isolated and purified the target antimicrobial peptides Salivaricin B, Salivaricin A2, BB, BA2, A2B, and A2A2.

[0040] The results showed that the antibacterial activity of the small peptide Salivaricin A2 alone was not as good as that of Salivaricin B. Except for the tandem peptide A2A2, which had no antibacterial activity, the other tandem peptides exhibited better antibacterial effects than the individual small peptides. The minimum inhibitory concentration (MIC) of the BB and A2B tandem peptides against *Streptococcus griseus* was 200 ng / μL; the MIC of the BB, A2B, and BA2 tandem peptides against *Streptococcus sanguinis* was 200 ng / μL; and the BB and A2B tandem peptides showed antibacterial effects against *Streptococcus mutans* at 300 ng / μL.

[0041] The present application will now be described in detail with reference to specific embodiments and accompanying drawings. These embodiments are for illustrative purposes only and should not be construed as limiting the scope of the application.

[0042] Example 1

[0043] 1 Experimental Methods

[0044] 1.1 Target Sequence Synthesis

[0045] The original nucleotide sequences of the Salivaricin B and Salivaricin A2 genes of Streptococcus salivarius strain K12 were obtained from the NCBI Nucleotide database.

[0046] Original Salivaricin A2 gene nucleotide sequence:

[0047] Streptococcus salivarius plasmid pSsal-K12 SalA(salA2),SalM(salM),SalT(salT),SalX(salX),SalY(salY),SalK(salK),SalR(salR),S boK(sboK),SboR(sboR),SboA(sboA),SboM(sboM),SboT(sboT),SboF(sboF),SboE(sboE),and SboG(sboG)genes,complete cds; and unknown genes

[0048] GenBank: DQ889451.1

[0049] CDS 1..156

[0050] / gene="salA2"

[0051] / codon_start=1

[0052] / transl_table=11

[0053] / product="SalA"

[0054] / protein_id="ABI63628.1"

[0055] The original nucleotide sequence of the Salivaricin A2 gene is shown in SEQ ID NO.1.

[0056] SEQ ID NO.1:

[0057]

[0058] The synthesis of its protein product involves a series of modification processes: the original Salivaricin A2 protein sequence, edited from the original Salivaricin A2 gene nucleotide sequence, is ultimately transformed into the sequence of the final effector peptide. During this process, the signal peptide leader sequence is shown in SEQ ID NO.2, the precursor protein propeptide is shown in SEQ ID NO.3, and the final effector peptide sequence is shown in SEQ ID NO.4.

[0059] SEQ ID NO.2: MKNSKDILNNAIEVSEKELMEVAGG

[0060] SEQ ID NO.3: KRGTGWFATITDDCPNSVFVCC

[0061] SEQ ID NO.4: KRGTGWFATITDDAPNSVFVAA

[0062] Post-translation modification structure, such as Figure 11 and Figure 12 As shown.

[0063] Original Salivaricin B gene nucleotide sequence:

[0064] Streptococcus salivarius plasmid pSsal-K12 SalA(salA2),SalM(salM),SalT(salT),SalX(salX),SalY(salY),SalK(salK),SalR(salR),S boK(sboK),SboR(sboR),SboA(sboA),SboM(sboM),SboT(sboT),SboF(sboF),SboE(sboE),and SboG(sboG)genes,complete cds; and unknown genes

[0065] GenBank: DQ889451.1

[0066] CDS 1..171

[0067] / gene="sboA"

[0068] / codon_start=1

[0069] / transl_table=11

[0070] / product="SboA"

[0071] / protein_id="ABI63639.1"

[0072] The original nucleotide sequence of the Salivaricin B gene is shown in SEQ ID NO.5.

[0073] SEQ ID NO.5:

[0074]

[0075] The synthesis of its protein product involves a series of modification processes: the original Salivaricin B protein sequence, edited from the original Salivaricin B gene nucleotide sequence, is ultimately transformed into the sequence of the final effector peptide. During this process, the signal peptide leader sequence is shown in SEQ ID NO. 6, the precursor protein propeptide is shown in SEQ ID NO. 7, and the final effector peptide sequence is shown in SEQ ID NO. 8.

[0076] SEQ ID NO.6: MAKQQMNLVEIEAMNSLQELTLEELDNVLGA

[0077] SEQ ID NO.7: GGGVIQTISHECRMNSWQFLFTCCS

[0078] SEQ ID NO.8: GGGVIQTISHECRMNSWQFLFTCCS

[0079] Post-translation modification structure, such as Figure 13 As shown.

[0080] The subsequent design was based on the sequences of Salivaricin B, Salivaricin A2, Salivaricin BA2, and Salivaricin BB peptide effectors. Enzyme restriction sites were added to the target nucleic acid sequence according to the multiple cloning site on the subsequently constructed expression vector. BamHI (GGATCC)-XhoI (CTCGAG) restriction sites were added to the 5' and 3' ends of the sequence, and the nucleotide sequence of the target peptide was directly synthesized chemically at Anhui General Biotechnology Co., Ltd. Simultaneously, upstream and downstream primers were designed based on the start and stop codons of the subsequently constructed expression vector.

[0081] The gene encoding the final effector peptide of Salivaricin B is shown in SEQ ID NO.9.

[0082] SEQ ID NO.9:

[0083] 5'-GGTGGTGGAGTAATCCAAACCATTTCACACGAATGTCGCATGAACTCATGGCAGTTCTTGTTTACTTGTTGCTCT-3'

[0084] The gene encoding the final effector peptide of Salivaricin A2 is shown in SEQ ID NO.10.

[0085] SEQ ID NO.10:

[0086] 5'-AAAAGAGGTACAGGTTGGTTTGCAACTATTACTGATGACTGTCCAAACTCAGTATTCGTTTGTTGT-3'

[0087] The upstream primer (F) is the sequence shown in SEQ ID NO.11, and the downstream primer (R) is the sequence shown in SEQ ID NO.12.

[0088] SEQ ID NO.11: 5'-TCCCATGGATGTCCCCTATAC-3'

[0089] SEQ ID NO.12: 5'-GGCCGCTCGAGACTAGTTTA-3'

[0090] This experiment designed a tandem peptide of Salivaricin B and Salivaricin A2, with six amino acids (GSGGSG) linked between the peptides. Simultaneously, upstream and downstream primers were designed based on the start and stop codons of the subsequently constructed expression vector.

[0091] To facilitate expression in heterologous prokaryotes, the codon-optimized BB sequence is shown in SEQ ID NO.13, and the BA2 sequence is shown in SEQ ID NO.14.

[0092] SEQ ID NO.13:

[0093]

[0094] SEQ ID NO.14:

[0095]

[0096] The upstream primer (F) is the sequence shown in SEQ ID NO.15, and the downstream primer (R) is the sequence shown in SEQ ID NO.16.

[0097] SEQ ID NO.15: 5'-GGCTGGCAAGCCACGTTTGGTG-3'

[0098] SEQ ID NO.16: 5'-CCGGGAGCTGCATGTGTCAGAGG-3'

[0099] 1.2 Construction of Recombinant Plasmids

[0100] This example constructed six recombinant plasmids: pGEX-6P-1-sal B, pGEX-6P-1-sal A2, pGEX-6P-1-BA2, pGEX-6P-1-A2A2, pGEX-6P-1-A2B, and pGEX-6P-1-BB, as detailed below:

[0101] 1.2.1 Carrier Processing

[0102] The pGEX-6P-1 plasmid was subjected to double digestion with BamHI and XhoI. The reaction system was 30 μL: Plasmid 6 μL, BamHI 2 μL, 10×buffer H 3 μL, ddH2O 17 μL, and XhoI 2 μL.

[0103] The double enzyme digestion reaction conditions were: 37℃ for 60 min.

[0104] The double-digested plasmid was ligated with the synthesized target series. The reaction system was 10 μL: 1 μL of digested pGEX-6P-1 vector, 4 μL of digested fragment, 1 μL of T4 DNA Ligase, 2 μL of 5×T4 DNA Ligase buffer, and 2 μL of ddH2O.

[0105] Mix thoroughly and allow to bind at room temperature for 20 minutes.

[0106] Preparation of E. coli DH5α competent cells using the calcium chloride method: Freshly grown single colonies were inoculated into 2 mL of LB broth and cultured at 37°C for 16 h. 1 mL of the bacterial culture was transferred to 50 mL of LB broth and cultured at 37°C with shaking for 2 h. The bacterial culture was placed on ice for 20 min. The cells were centrifuged at 4000 rpm for 10 min at 4°C to collect the bacteria. The bacteria were resuspended in 10 mL of pre-chilled 30 mM CaCl2 solution (on ice), incubated on ice for 40 min, and then centrifuged at 4000 rpm for 10 min at 4°C. The bacterial pellet was resuspended in 400 μL of pre-chilled 30 mM CaCl2 solution (on ice) and aliquoted into sterile 1.5 mL EP tubes (120 μL / tube). The tubes were stored at -70°C for later use.

[0107] Transformation of E. coli DH5α competent cells with recombinant plasmid: Mix 1 μL of recombinant plasmid with 150 μL of freshly prepared E. coli DH5α competent cells and incubate on ice for 30 min. Heat shock in a 42℃ water bath for 90 s, then immediately place on ice for 2 min. In a clean bench, add 800 μL of antibiotic-free LB liquid medium and incubate at 37℃ on a shaker at 160 rpm for 1 h to allow recovery. Centrifuge at 5000 rpm for 5 min. In a clean bench, reserving 100 μL of supernatant, resuspend the bacterial cells by pipetting. In a clean bench, spread 100 μL of bacterial culture evenly onto LB solid medium containing 100 μg / mL AMP. Incubate at room temperature for 10-20 min until absorption, then invert the plate and incubate at 37℃ for 12-16 h until colonies appear.

[0108] 1.2.2 Plasmid extraction and sequencing identification

[0109] Four single colonies from LB agar were inoculated into 4 mL of LB liquid medium containing 100 μL / mL AMP and incubated overnight at 37°C with shaking. Plasmids were extracted using the Novizan plasmid kit. Restriction sites were predicted using software. For pGEX-6P-1-sal B, EcoRV-XhoI was used for double digestion; for pGEX-6P-1-sal A2, ApaI was used; for pGEX-6P-1-BA2, ScaI was used; for pGEX-6P-1-A2A2, ScaI was used; for pGEX-6P-1-A2B, ScaI was used; and for pGEX-6P-1-BB, ScaI was used. The reaction mixture consisted of 10 μL of recombinant plasmid, 1 μL of 10× buffer H, 0.5 μL of restriction enzyme, and 7.5 μL of H2O. The reaction was carried out at 37°C for 3 hours. After adding 10× Loading buffer, 4 μL of the solution was taken for 1% agarose gel electrophoresis to detect the size of the enzyme digestion fragments.

[0110] 1.3 Construction of cloning vectors

[0111] 1.3.1 Preparation of E. coli DH5α competent cells (calcium chloride method):

[0112] ① Take a freshly grown single colony and inoculate it into 2 mL of LB liquid medium, and incubate at 37℃ for 16 h.

[0113] ② Transfer 1 mL of bacterial culture to 50 mL of LB medium and incubate at 37°C with shaking for 2 h.

[0114] ③ Place the bacterial culture on ice for 20 minutes.

[0115] ④ Centrifuge at 4℃, 4000 rpm / min, for 10 min, and collect the bacteria.

[0116] ⑤ Suspend the bacteria in 10 mL of 30 mM CaCl2 solution pre-cooled on ice, incubate on ice for 40 min, then centrifuge at 4000 rpm for 10 min at 4 °C.

[0117] ⑥ Resuspend the bacterial pellet in 400 μL of ice-cold 30 mM CaCl2 solution, and aliquot the bacteria into sterile 1.5 mL EP tubes, 120 μL / tube. Store at -70°C for later use.

[0118] 1.3.2 Transformation of recombinant plasmids into E. coli DH5α competent cells

[0119] ① Mix 1 μL of recombinant plasmid with 150 μL of freshly prepared E.coli DH5α competent cells and incubate on ice for 30 min.

[0120] ② Heat shock in a 42℃ water bath for 90 seconds, then quickly place on ice for 2 minutes.

[0121] ③ Perform the procedure in a clean bench, add 800 μL of antibiotic-free LB liquid medium, incubate at 37°C in a shaker at 160 rpm / min, and allow to recover for 1 h.

[0122] ④ Centrifuge at 5000 rpm for 5 min.

[0123] ⑤ Perform the procedure in a clean bench, retain 100 μL of supernatant, and resuspend the bacterial cells by pipetting.

[0124] ⑥ Perform the operation in a clean bench, take 100 μL of bacterial culture and spread it evenly on LB solid medium containing 100 μg / mL AMP.

[0125] ⑦ Place at room temperature for 10-20 minutes to allow absorption, then invert the plate and incubate at 37℃ for 12-16 hours until colonies appear.

[0126] 1.3.3 Plasmid extraction and sequencing identification using the Novizan kit

[0127] ①The following day, single clones were selected from LB plates and inoculated into test tubes containing 100 μg / ml AMP in LB liquid medium and cultured at 37°C and 160 rpm / min.

[0128] ② Take 1 mL of fresh bacterial culture containing recombinant plasmid into a sterile 1.5 mL centrifuge tube.

[0129] ③ Centrifuge at 12000 rpm for 1 min and remove the supernatant.

[0130] ④ Extract plasmids using the Novizan reagent kit and send them to Anhui General Biotechnology Co., Ltd. for sequencing.

[0131] 1.4 Constructing expression vectors

[0132] 1.4.1 Preparation of E. coli BL21(DE3) competent cells (calcium chloride method):

[0133] The preparation of competent cells is the same as that for E. coli DH5α competent cells (calcium chloride method).

[0134] 1.4.2 Transformation of recombinant plasmid into E. coli BL21(DE3) competent cells:

[0135] The transformation of the recombinant plasmid was the same as that of E. coli DH5α competent cells.

[0136] 1.5 Expression identification, optimization and solubility analysis

[0137] 1.5.1 Expression and Identification of Antimicrobial Peptides

[0138] (1) The clones were inoculated into 5 tubes of LB medium containing 100 μg / mL AMP resistance and cultured at 37°C for 16 hours to preserve the bacteria. 300 μL of fresh bacterial culture was added to a cryovial containing 200 μL of 50% glycerol that had been autoclaved beforehand.

[0139] (2) Inoculate the bacterial cells into 5 tubes of LB medium containing 100 μg / mL AMP resistance and incubate at 37°C until the bacterial cells reach OD. 600 The concentration was 0.6-0.8 (approximately 3 hours). IPTG was added to a final concentration of 0.5 mM. After culturing for 4 hours, the bacteria were collected by centrifugation at 5000g for 10 minutes at 4°C.

[0140] (3) Take a small amount of bacterial precipitate and put it into a sterile 0.5 mL EP tube. Add 30 μL of sterile water and 6 μL of 5×SDS loading buffer. Boil in boiling water for 10 min. Load the sample for SDS-PAGE and Western Blot analysis.

[0141] (4) Prepare SDS-PAGE gels. Individual small peptides are approximately 30 kDa in size, and tandem peptides are approximately 33 kDa in size. Prepare a 15% separating gel (one gel): 1200 μL ddH2O; 2500 μL 30% Acr / Bis; 1250 μL 4× separating gel; 50 μL 10% AP; 5 μL TEMED. Prepare a 6% stacking gel (one gel): 1350 μL ddH2O; 500 μL 30% Acr / Bis; 625 μL 4× stacking gel; 50 μL 10% AP; 5 μL TEMED.

[0142] (5) Western Blot Analysis. First, remove excess material from the SDS-PAGE gel. Then, cut one nitrocellulose membrane (NC membrane) and six sheets of filter paper to the same size as the gel strip, and thoroughly soak them in transfer buffer. In a semi-dry electrotransfer tank, stack three sheets of filter paper from the anode to the cathode. One layer of NC membrane, one layer of gel, three layers of filter paper, remove any air bubbles with a glass rod, and transfer at room temperature for 0.5 hours. Place the NC membrane in a pre-prepared PBST buffer containing 5% skim milk powder and block at room temperature for 2 hours. Wash the membrane three times with PBST buffer for 10 minutes each time. Then, place the NC membrane in HRP-Conjugated GST Tag Mouse McAb diluted 1:10000 with PBST, incubate at 37°C for 0.5 hours, incubate at room temperature for 1.5 hours, wash the membrane three times with PBST buffer for 10 minutes each time, and develop the product with DAB.

[0143] 1.5.2 Expression optimization and solubility analysis of antimicrobial peptides

[0144] (1) Inoculate the bacterial cells into 4 tubes of LB medium containing 100 μg / mL AMP resistance and incubate at 37°C until the bacterial cell OD reaches 100 μg / mL. 600 The concentration was 0.6-0.8 (approximately 3 hours). IPTG was added to final concentrations of 0.2 mM and 1 mM, respectively, and the mixture was cultured at 37℃ and 15℃ at 220 rpm for 4 hours and 16 hours, respectively, to induce fusion protein expression.

[0145] (2) Transfer the well-grown bacterial culture to centrifuge tubes, centrifuge at 5000g for 10 min to collect the bacteria, and resuspend the bacteria in PBS buffer containing a final concentration of 5 mM DTT. The bacterial concentration should be moderate, and the entire process should be performed on ice. Sonicate the bacteria for 15 min, with 30 s working and 30 s rest intervals, at a power of 38%-40%.

[0146] (3) After the ultrasonically disrupted bacterial solution was centrifuged at 10000g for 30 min at 4℃, it was separated into supernatant and precipitate. The precipitate was mixed by blowing with PBS. Then, the supernatant and precipitate were prepared separately. 30 μL of each was injected into a 0.5 Ml EP tube, 6 μL of 5×SDS loading buffer was added, and the tube was boiled in boiling water for 10 min. The tube was then loaded for SDS-PAGE analysis.

[0147] 1.6 Isolation and purification of the target protein

[0148] 1.6.1 Expression of target protein

[0149] (1) Select single clones from LB plates and inoculate them into LB liquid medium containing 100 μg / mL AMP in test tubes and incubate at 37°C and 160 rpm / min.

[0150] (2) Take 1 mL of fresh bacterial culture from the test tube and add it to an Erlenmeyer flask containing 100 μg / mL AMP in LB liquid medium. Shake the flask at 37°C and 160 rpm / min.

[0151] (3) Cultivate to bacterial OD 600 Add IPTG to a final concentration of 0.5 mM (approximately 0.6-0.8 mM) after 4 hours of incubation. Centrifuge at 10000g, 4°C for 10 minutes, discard the supernatant, and collect the precipitate.

[0152] 1.6.2 Protein Purification

[0153] (1) Ultrasonic lysis: Bacterial cells were lysed by sonication using 5mM DTT pH 7.0 PBS buffer;

[0154] (2) Centrifugation: 10000g, centrifuge at 4℃ for 30min;

[0155] (3) Mix the supernatant after centrifugation with the affinity resin column at 15°C and shake for two hours.

[0156] (4) After reloading the column, let it stand and separate into layers, allowing the supernatant to flow out of the column.

[0157] (5) Washing miscellaneous items:

[0158] Washing conditions for individual small peptides:

[0159] a. Wash with PBS at pH 7.0 for approximately 10 column volumes;

[0160] b. Wash with PBS-0.5M NaCl at pH 7.0 for approximately 5 column volumes;

[0161] c. Wash with PBS-1M NaCl at pH 7.0 for approximately 5 column volumes;

[0162] d. Rinse with deionized water for 3 column volumes, and take the washed medium for SDS-PAGE.

[0163] Washing conditions for tandem peptides:

[0164] a. Wash approximately 10 column volumes with 0.3% Triton X 100 pH 7.0 PBS;

[0165] b. Wash with PBS-0.5M NaCl at pH 7.0 for approximately 5 column volumes;

[0166] c. Wash with PBS-1M NaCl at pH 7.0 for approximately 5 column volumes;

[0167] d. Wash with PBS-1.5M NaCl at pH 7.0 for approximately 5 column volumes.

[0168] e. Rinse with deionized water for 3 column volumes, and take the washed medium for SDS-PAGE.

[0169] (6) Elution: Elute with 50mM Tris-HCl and 10mM reduced glutathione at pH 8.0 for 15 column volumes;

[0170] (7) After elution, wash the packing material with 5 column volumes of PBS and 5 column volumes of deionized water in sequence, and finally wash with 3 column volumes of 20% ethanol. The ethanol should completely submerge the packing material. After sealing the column, store at 4°C.

[0171] 1.6.3 SDS-PAGE Analysis

[0172] (1) Centrifuge the ultrasonically lysed bacterial culture and use the supernatant for column loading. Before column loading, take 30 μL for SDS-PAGE to verify the protein content of small peptides in the supernatant.

[0173] (2) Use 5 mL of deionized water to blow and centrifuge the precipitate, and take 30 μL for SDS-PAGE to verify the protein content of small peptides in the precipitate.

[0174] (3) Take 30 μL of elution buffer 1 and perform SDS-PAGE to observe the elution of the target protein and other proteins.

[0175] (4) Take 30 μL of the elution resin solution of elution buffer 2 and perform SDS-PAGE to observe the elution of impurities.

[0176] (5) Take 30 μL of the washed resin column and perform SDS-PAGE to observe the content of the target protein and the content of impurity proteins;

[0177] (6) Take 30 μL of the eluted liquid and perform SDS-PAGE to determine if it contains the target protein.

[0178] 1.6.4 Amino acid sequencing

[0179] (1) Cut the target strip from the SDS-PAGE obtained in 2.6.3 with a clean blade, removing as much of the excess as possible.

[0180] (2) Place the gel into a clean 1.5mL centrifuge tube and label it.

[0181] (3) Add 1 mL of water to each centrifuge tube, wash for 10 minutes, remove the water, and repeat once. Add 1 mL of in-gel digestion and decolorization buffer to each centrifuge tube, wash for 10 minutes, remove the decolorization buffer, and repeat once. Preparation of in-gel digestion and decolorization buffer: 50% acetonitrile, 25 mM ammonium bicarbonate.

[0182] (4) Add acetonitrile to dehydrate until the granules turn completely white, and then vacuum dry the acetonitrile.

[0183] (5) Add 10mM DTT to allow the particles to fully absorb the DTT, then place the mixture in a 56℃ water bath and incubate for 1 hour.

[0184] (6) After incubation, remove excess DTT liquid, add 55mM IAM, and incubate in a dark room at room temperature for 45 minutes.

[0185] (7) After incubation, remove excess IAM liquid, add 25mM ammonium bicarbonate, wash for 10 minutes, and repeat the washing once.

[0186] (8) Remove ammonium bicarbonate, add decolorizing solution and wash for 10 minutes, and repeat once.

[0187] (9) Dehydrate acetonitrile until the granules turn completely white, then vacuum dry the acetonitrile.

[0188] (10) Dilute the 1 μg / μL enzyme stock solution 15 times with 25 mM ammonium bicarbonate and add it to the dehydrated gel particles, allowing the particles to fully absorb it. Then add 25 mM ammonium bicarbonate to cover the gel particles and place them in a 37°C water bath for overnight digestion.

[0189] (11) After overnight digestion, add FA to a final concentration of 0.1% to terminate digestion.

[0190] (12) Take 10 μL of sample and put it on the instrument for mass spectrometry.

[0191] 2 Results

[0192] 2.1 Results of Recombinant Plasmid Detection

[0193] The results showed that the Salivaricin B, Salivaricin A2, A2B, BA2, A2A2, and BB genes were successfully introduced into the pGEX-6p-1 plasmid.

[0194] The pGEX-6p-1 plasmid contains a GST tag. GST increases the solubility of the target protein, and during purification, the GST tag is used for affinity chromatography with a GST resin column, enabling efficient and rapid separation of the target antimicrobial peptide. The Salivaricin B gene is 75 bp in size, the Salivaricin A2 gene is 66 bp in size, and the BA2 gene is 159 bp in size. The electrophoresis results of the recombinant plasmids are as follows: Figure 1 As shown. Figure 1Lane 7 contains the pGEX-6P-1-BB recombinant plasmid; lane 8 contains the pGEX-6P-1-BB recombinant plasmid digested with ScaI; lane 9 contains the pGEX-6P-1-BA2 recombinant plasmid; lane 10 contains the pGEX-6P-1-BA2 recombinant plasmid digested with ScaI; lane 11 contains the pGEX-6P-1-BA2 recombinant plasmid; lane 12 contains the pGEX-6P-1-BA2 recombinant plasmid digested with ScaI; lane M is the DNA Marker.

[0195] Therefore, the size of a single small peptide is approximately 30 kDa, and the size of a tandem peptide is approximately 33 kDa. To investigate the biological activity of single small peptides and tandem peptides, this experiment purchased the pET-42a plasmid, which encodes the GST protein, as a negative control protein targeting the peptide.

[0196] The results of SDS-PAGE and Western blot analysis of the target protein expression in E. coli BL21(DE3) strain are as follows: Figure 2 As shown, af: SDS-PAGE and Western blot analyses of Salivaricin B, Salivaricin A2, Salivaricin BA2, Salivaricin BB, Salivaricin A2A2, and Salivaricin A2B peptides, respectively, where 1 represents uninduced expression; 2-6 represent induced expression; and M represents Protein Marker.

[0197] The target gene sequence was obtained through direct synthesis. The synthesized target fragment was ligated into the pGEX-6p-1 plasmid and transformed into E. coli DH5α competent cells. Single colonies from solid plates were picked for liquid culture, and then the plasmid was extracted. pGEX-6p-1-BA2 and pGEX-6p-1-BB were digested with the restriction endonuclease ScaI for identification. The digestion results were correct. Figure 1 As shown in the figure, the correctly digested plasmid was sent to Anhui General Biotechnology Co., Ltd. for sequencing. The sequenced nucleotide sequence was compared with the gene sequence of the recombinant target peptide. The results showed that the sequences were consistent, indicating that there were no mutations and that the gene and protein were correct.

[0198] The correctly sequenced plasmid was transformed into E. coli BL21(DE3) competent cells. Single colonies were picked and transferred to LB liquid medium containing 100 μg / ml AMP. By comparing cells without IPTG with those containing 0.5 mM IPTG, the target protein was found to be highly expressed under IPTG induction. Western blot results showed a GST-tagged protein at approximately 30 kDa. Figure 2As shown above, the experimental results indicate that the target protein was successfully expressed in E. coli BL21(DE3) strain.

[0199] 2.2 Expression identification, optimization, and solubility analysis of the target protein

[0200] The results of expression identification, optimization, and solubility analysis of the tandem peptides are as follows: Figure 3 As shown. Figure 3 Figure a shows the expression identification, optimization, and solubility analysis of BA2; Figure b shows the expression identification, optimization, and solubility analysis of BB; M: Protein Marker; 1: Sample induced by 0.2mM IPTG at 15℃; 2: Sample induced by 1.0mM IPTG at 15℃; 3: Sample induced by 0.2mM IPTG at 37℃; 4: Sample induced by 1.0mM IPTG at 37℃; 5: Uninduced sample; 6: Precipitate after induction by 1.0mM IPTG at 37℃; 7: Supernatant after induction by 1.0mM IPTG at 37℃; 8: Precipitate after induction by 0.2mM IPTG at 37℃; 9: Supernatant after induction by 0.2mM IPTG at 37℃; 10: Precipitate after induction by 1.0mM IPTG at 15℃; 11: Supernatant after induction by 1.0mM IPTG at 15℃; 12: Precipitate after induction by 0.2mM IPTG at 15℃. Precipitated sample after IPTG induction; Supernatant sample after IPTG induction at 13:15℃ and 0.2mM.

[0201] Figure 3 The results showed that the expression levels of the tandem peptides BA2 and BB were highly dependent on the induction temperature. At 37℃, the expression levels of the tandem peptides were relatively low, and the content of the target protein in the supernatant was even lower. Therefore, an induction temperature of 15℃ and 16 h was chosen. At 15℃, there was no significant difference in expression levels between 0.2 mM and 1 M IPTG concentrations; therefore, an intermediate concentration of 0.5 mM was selected. In summary, the induction conditions for the tandem peptides BA2 and BB are: 0.5 mM IPTG, 15℃, and 16 h of shake-flask culture.

[0202] 2.3 Isolation and purification of target protein

[0203] The target protein was isolated and purified by designing NaCl concentration gradients of 0, 0.5M, 1M, and 1.5M in PBS buffer, pH gradients of 7.0 and 8.0, and Triton X 100 detergent concentration gradients of 0 and 0.3%. Experimental results showed that the washing and elution conditions for the tandem peptides BA2 and BB were as follows: approximately 10 column volumes of PBS-0.3% Triton X 100-pH 7.0; approximately 5 column volumes of PBS-0.5M NaCl-pH 7.0; approximately 5 column volumes of PBS-1M NaCl-pH 7.0; approximately 5 column volumes of PBS-1.5M NaCl-pH 7.0; and elution with 50mM Tris-HCl-10mM reduced glutathione-pH 8.0 for 10-15 column volumes. Figure 4 As shown.

[0204] Figure 4 The following are the SDS-PAGE analysis results for the separation and purification of BA2, BB, A2A2, and A2B. Figure a shows the SDS-PAGE analysis of BA2 separation and purification; Figure b shows the SDS-PAGE analysis of BB separation and purification. Wherein, M: Protein Marker; 1: Bacterial sonication and precipitation; 2: Bacterial sonication supernatant; 3: Eluent from the resin column after the bacterial sonication supernatant has been bound to the resin column at 15℃ for 2 hours; 4: Washing solution; 5: Purified protein; S: After optimization, four washing steps were finally determined: 10 column volumes of PBS-0.3% Triton X 100-pH 7.0, 5 column volumes of PBS-0.5M NaCl-pH 7.0, 5 column volumes of PBS-1M NaCl-pH 7.0, and 5 column volumes of PBS-1.5M NaCl-pH 7.0, yielding the purified protein.

[0205] 2.4 Amino acid mass spectrometry analysis

[0206] Mass spectrometry data were analyzed using Data Analysis Software. The amino acid sequences were obtained through a data search using Mascot searchengine version 2.3.01. The amino acid sequencing results were then compared with those obtained using the protein alignment method in NCBI's BLAST (https: / / blast.ncbi.nlm.nih.gov / BlastAlign.cgi). The results showed that the amino acid sequencing results for BA2 and BB were consistent with expectations.

[0207] Example 2

[0208] 1 Experimental Methods

[0209] 1.1 Bacterial Culture

[0210] Three cariogenic streptococci and one novel strain HC61B-CBA-3-1 were identified from the primary oral flora. Frozen strains were removed from a -80°C freezer, and the revived glycerol culture was streaked onto GAM anaerobic solid medium in an anaerobic incubator. This medium, along with the anaerobic bag, was placed in an anaerobic incubator and incubated for approximately 24 hours to form single colonies. Single colonies were picked and inoculated into GAM liquid anaerobic tubes. Streptococci grew for 16 hours, and HC61B-CBA-3-1 grew for 37 hours, with monitoring to prevent contamination throughout the process.

[0211] 1.2 Protein Quantification

[0212] 1.2.1 The isolated and purified antimicrobial peptides were concentrated using Centrifugal filters (10000 MWCO), centrifuged at 4°C and 4000g for 5 min, and the supernatant was retained. Then, PBS buffer was added, and centrifugation continued. The eluent was washed five times with PBS to remove reduced glutathione. When using BCA for protein quantification, reduced glutathione can affect the protein concentration measurement, leading to a higher measured concentration than the actual concentration.

[0213] 1.2.2 BCA protein concentration kit. Under alkaline conditions, proteins will convert Cu... 2+ Reduced to Cu + Cu + It interacts with the unique BCA Reagent A (containing BCA) to produce a sensitive color reaction, forming a purple complex. This water-soluble complex in A... 562 It exhibits strong absorbance at nm, and the absorbance shows a good linear relationship with protein concentration over a wide range. Protein concentration can be calculated from the absorbance value. Therefore, it is suitable for measuring its concentration at A nm using a microplate reader. 562 The concentration of the protein to be tested can be calculated by comparing the absorbance value at nm with the standard curve.

[0214] 1.2.3 Instructions for using the BCA protein concentration kit:

[0215] (1) Preparation of BCA working solution. Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA Reagent B to 50 volumes of BCA Reagent A (50:1) and mix thoroughly.

[0216] (2) Plotting the standard curve. Take an ELISA plate and add reagents according to the data in the table below.

[0217] Table 1 Standard Curve Samples

[0218] Protein standard (μL) 0 1 2 4 8 12 16 20 Deionized water (μL) 20 19 18 16 12 8 4 0 BCA working solution (μL) 200 200 200 200 200 200 200 200 Corresponding protein content (μg) 0 1 2 4 8 12 16 20

[0219] (3) Sample preparation: Dilute the protein sample to be tested with deionized water to an appropriate concentration, take 20 μL of sample and add 200 μL of BCA working solution.

[0220] (4) After shaking and mixing, place at 37°C for 30 min.

[0221] (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at 562 nm. Use the absorbance without BSA as a blank control.

[0222] (6) Plot a standard curve with protein content (μg) on ​​the x-axis and absorbance on the y-axis.

[0223] (7) The protein content of the sample can be calculated from the standard curve based on the measured absorbance value.

[0224] (8) Calculate the protein concentration: Divide the protein content found by the sample volume of 20 μL, and then multiply by the corresponding dilution factor to obtain the actual reading of the sample to be tested.

[0225] 1.3 Antibacterial test

[0226] 1.3.1 AlamarBlue test of antimicrobial peptides against cariogenic bacteria and HC61B-CBA-3-1

[0227] (1) Preparation of resazurin colorimetric solution: First, dissolve resazurin powder in ddH2O to a concentration of 12.5 mg / mL as a high-concentration stock solution and store at 4℃; take 10 mL of Tween-80 and add it to 90 mL of ddH2O, autoclave, and mix immediately after taking it out of the autoclave; take 1 mL of the high-concentration resazurin stock solution and add it to 99 mL of ddH2O and mix thoroughly; filter the diluted resazurin through a 0.22 μm filter to sterilize, and add the filtrate to the sterilized 10% Tween-80. This is the resazurin working solution, which should be stored at 4℃ and used within one week.

[0228] (2) Specific operation: In the anaerobic chamber, resuscitate streptococci and HC61B-CBA-3-1, dilute the bacterial solution 1000 times before use, and use the concentrated antimicrobial peptide concentration as the working concentration. After dilution according to a certain serial dilution method, add it to a 96-well plate with a total volume of 100 μL. Make up the difference with sterile GAM. After incubating in the anaerobic chamber at 37℃ for 4 hours, add 100 μL of resazurin working solution per well and continue to incubate and observe.

[0229] To refine the razor cyanide assay, blank control, positive control, negative control, and antibiotic control were designed. The experiment is as follows:

[0230] ① Blank control: 100 μL GAM medium to confirm that GAM does not affect the color change of rezamidophos.

[0231] ② Negative control: GST protein and bacterial cell dilution to exclude the influence of GST protein on bacterial cells.

[0232] ③ Negative control: small peptide and GAM, to confirm that the small peptide does not affect the color change of azure.

[0233] ④ Antibiotic control: AMP / Kan, bacterial cell dilution, to confirm that the bacteria can indeed be inhibited.

[0234] ⑤ Positive control: Bacterial cell dilution, with free bacterial growth, to test the antibacterial effect of the small peptide.

[0235] ⑥ 500 ng / μL small peptide, bacterial cell dilution solution

[0236] ⑦ 1000 ng / μL small peptide, bacterial cell dilution solution

[0237] ⑧ 2000 ng / μL small peptide, bacterial cell dilution solution

[0238] 1.3.2 Growth curves of cariogenic bacteria and HC61B-CBA-3-1 under the action of antimicrobial peptides

[0239] Antimicrobial peptides were added to fresh liquid anaerobic culture medium to a final concentration of 250 μg / mL. Simultaneously, the cultured bacterial suspension was inoculated into the liquid culture medium at a ratio of 1:500. Based on the growth curves of various streptococci and HC61B-CBA-3-1, OD values ​​were measured at specific points. 600 Numerical values ​​were used to plot growth curves. To detect the number of viable bacteria at different time points under the action of the antimicrobial peptide, bacterial solutions were taken at corresponding time points and serially diluted 10-fold. The OD values ​​were then used to determine the growth rate. 600 Dilute the culture to an appropriate factor, take 10 μL and inoculate it onto GAM solid anaerobic medium, place it in an anaerobic bag, and incubate at 37°C. Incubate Streptococcus for about 16 hours, then incubate HC61B-CBA-3-1 for 36 hours. Count the number of colonies and plot the results.

[0240] 2 Results

[0241] 2.1 Results of the Azure Blade Experiment

[0242] A micro-inhibition experiment was conducted using 96-well plates. The different colors displayed by the redox reaction of resazurin served as indicators. Resazurin appears blue in its reduced state, and undergoes an irreversible color change from blue to pink when oxidized. Finally, it becomes colorless as the redox potential increases. In the micro-inhibition experiment, if there was no bacterial growth in the 96-well plate, the resazurin was blue; the greater the bacterial count, the more the resazurin transitioned from blue to pink and then to colorless. The color reaction directly reflects the amount of bacteria in the 96-well plate.

[0243] In the antimicrobial assay of 96-well plates, three common cariogenic streptococci in the oral cavity were selected. The reason for selecting these three strains is that antimicrobial peptides inhibit the growth of strains of the same genus as the antimicrobial peptide-producing strains. At the same time, interactions between various streptococci can be observed in the isolation and culture of oral flora. Therefore, streptococci were the first choice when selecting strains that indicate antimicrobial peptide activity.

[0244] When isolating and culturing oral flora in 330 medium, colony interactions can be clearly observed in 330 medium, including Streptococcus griseus, Streptococcus sanguinis, etc.

[0245] In the 96-well plate antibacterial experiment, blank control, negative control and positive control were designed, as well as antibacterial peptide concentration gradients, in order to determine the minimum inhibitory concentration through different concentration gradients.

[0246] In the *Streptococcus griseus* inhibition experiment, the bacterial dilution solution turned red, while the blank control showed no change, indicating that bacterial growth caused resamaritan to turn red. In the four concentration gradients of Salivaricin A2 and Salivaricin B, resamaritan turned red, but at 1000 ng / μl and 2000 ng / μl, it remained bluish, indicating its antibacterial activity. The total duration of the small peptide inhibition experiment was 7 hours, and the antibacterial effect was not ideal; *Streptococcus griseus* was not completely inhibited, but the antibacterial effect of Salivaricin B was better than that of Salivaricin A2.

[0247] In the tandem antimicrobial peptide inhibition assay for *Streptococcus griseus*, A2A2 showed no antimicrobial activity. At 100 ng / μL, only A2B showed antimicrobial activity, while the other three antimicrobial peptides had no effect. At 200 ng / μL, all peptides except A2A2 showed antimicrobial activity, with BB and A2B peptides completely inhibiting *Streptococcus griseus* growth, indicating that the minimum inhibitory concentration (MIC) of BB and A2B peptides against *Streptococcus griseus* was 200 ng / μL. At 300 ng / μL, BB and A2B peptides completely inhibited *Streptococcus griseus* growth. In the interaction of small peptides with resazurin, resazurin did not turn red, indicating that small peptides did not affect the resazurin assay results. In bacterial suspensions containing GST protein, resazurin turned colorless, and comparison with the color of bacterial suspensions without GST protein showed that GST protein promotes *Streptococcus griseus* growth. The total duration of the tandem peptide antibacterial experiment was 21 hours. At this point, AMP had turned red, consistent with the principle that AMP is easily decomposed by heat and loses its antibacterial properties. Kan turned slightly red, indicating that the BB and A2B tandem peptides at 200 ng / μL had better antibacterial effects than 100 ng / μL AMP and 50 ng / μL Kan. By extending the experimental time, it was found that the BB and A2B antimicrobial peptides still had antibacterial properties even after more than 50 hours, indicating that these two antimicrobial peptides have thermal stability.

[0248] The antibacterial experiments of Salivaricin A2 and Salivaricin B against Streptococcus griseus, and the control design are shown in Table 2.

[0249] Table 2. Composition of each well in the 96-well plate experiment.

[0250] A Bacterial cell dilution Bacterial cell dilution Bacterial cell dilution blank blank blank B 100ng / μLA2 100ng / μLA2 100ng / μLA2 100ng / μLB 100ng / μLB 100ng / μLB C 500ng / μLA2 500ng / μLA2 500ng / μLA2 500ng / μLB 500ng / μLB 500ng / μLB D 1000ng / μLA2 1000ng / μLA2 1000ng / μLA2 1000ng / μLB 1000ng / μLB 1000ng / μLB E 2000ng / μLA2 2000ng / μLA2 2000ng / μLA2 2000ng / μLB 2000ng / μLB 2000ng / μLB F A2, GAM A2, GAM A2, GAM B, GAM B, GAM B, GAM G Elution solution Elution solution Elution solution Elution solution Elution solution Elution solution H AMP AMP AMP kan kan kan

[0251] The antibacterial experiments of BB, BA2, A2A2 and A2B against Streptococcus griseus, as well as the composition of each well in the 96-well plate experiment, are shown in Table 3.

[0252] Table 3. Composition of each well in the 96-well plate experiment.

[0253] A blank Bacterial cell dilution B 100ng / μLBB 100ng / μLBA2 100ng / μLA2A2 100ng / μLA2B C 200ng / μLBB 200ng / μLBA2 200ng / μLA2A2 200ng / μLA2B D 300ng / μLBB 300ng / μLA2A2 300ng / μLA2B E BB, GAM BA2, GAM A2A2, GAM A2B, GAM F GST protein G 100 ng / μLAMP H 50ng / μL kan

[0254] In the Streptococcus sanguinis inhibition experiment, the bacterial cell dilution solution turned red, while the blank control showed no change, indicating that bacterial growth caused the resalicylate to turn red. At 1000 ng / μL and 2000 ng / μL, the resalicylate remained at a bluish level, indicating effective inhibition of Streptococcus sanguinis. At 500 ng / μL and 1000 ng / μL, the resalicylate remained at a bluish level, indicating that at this concentration, Salivaricin B had an inhibitory effect on Streptococcus sanguinis, and at 2000 ng / μL, Streptococcus sanguinis was severely inhibited. The total duration of the single small peptide inhibition experiment was 7 hours. In the four inhibitory concentration gradients, both Salivaricin A2 and Salivaricin B showed inhibitory effects, with Salivaricin B showing a better inhibitory effect than Salivaricin A2, but complete inhibition of Streptococcus sanguinis was not observed in either experiment.

[0255] Table 2 shows the antibacterial experiments of Salivaricin A2 and Salivaricin B against Streptococcus sanguinis, as well as the composition of each well in the 96-well plate experiment.

[0256] Table 3 shows the antibacterial experiments of BB, BA2, A2A2 and A2B against Streptococcus sanguinis, as well as the composition of each well in the 96-well plate experiment.

[0257] In the tandem antimicrobial peptide inhibition assay for *Streptococcus halophilus*, A2A2 showed no antimicrobial activity. At 100 ng / μL, all three tandem peptides except A2A2 showed slight antimicrobial effects. At 200 ng / μL, except for A2A2, BB, A2B, and BA2 antimicrobial peptides completely inhibited the growth of *Streptococcus halophilus*, therefore the minimum inhibitory concentration (MIC) of these three antimicrobial peptides was 200 ng / μL. No color change occurred when the antimicrobial peptides reacted with resazurin, indicating that small peptides do not affect the resazurin assay results. In bacterial cultures containing GST protein, resazurin turned red, indicating that GST protein cannot inhibit the growth of *Streptococcus halophilus*. The total duration of the tandem peptide inhibition assay was 21 hours. At this time, AMP turned red, consistent with the principle that AMP is easily decomposed by heat and loses its antimicrobial activity. Kan showed no color change, indicating that 50 ng / μL Kan completely inhibited the growth of *Streptococcus griseus*. By extending the experimental time, it was found that the antimicrobial peptides BB, BA2, and A2B still exhibited antibacterial activity after more than 50 hours, indicating that these three antimicrobial peptides possess thermal stability.

[0258] In the Streptococcus mutans inhibition experiment, the bacterial dilution turned red, while the blank control showed no change, indicating that bacterial growth caused the resalivaricin to turn red; Salivaricin A2 and Salivaricin At concentrations of 1000 ng / μL and 2000 ng / μL, resamaritan turned completely red, indicating that the single peptide had no antibacterial activity against Streptococcus mutans. Among the tandem antimicrobial peptides, A2A2 and BA2 showed no antibacterial activity, but BA2 was lighter in color than A2A2, indicating that BA2 had a higher bacterial concentration than A2A2. At concentrations of 200 ng / μL and 300 ng / μL, both BB and A2B remained slightly blue, thus showing a slight antibacterial effect. The blue color of BB peptide was deeper than that of A2B peptide, indicating that BB peptide had better antibacterial activity than A2B. In the interaction of small peptides with resamaritan, resamaritan did not turn red, indicating that small peptides did not affect the results of the resamaritan experiment. In bacterial solutions containing GST protein, resamaritan turned red, indicating that GST protein could not inhibit the growth of Streptococcus mutans. In this experiment, both AMP and Kan turned red, indicating that 100 ng / μL AMP and 50 ng / μL Kan could not inhibit the growth of Streptococcus mutans. This further demonstrates that 200 ng / μL BB peptide and 300 ng / μL A2B peptide had stronger antibacterial effects than 100 ng / μL AMP and 50 ng / μL Kan. The total duration of the tandem peptide antibacterial experiment was 21 hours. While BB and A2B antimicrobial peptides inhibited the growth of Streptococcus mutans, complete inhibition was not observed, so the minimum inhibitory concentration could not be determined.

[0259] Table 4 shows the antibacterial experiments of Salivaricin A2, Salivaricin B, BB, BA2, A2A2 and A2B against Streptococcus mutans, as well as the composition of each well in the experiment.

[0260] Table 496 describes the composition of each well in the 6-well plate experiment.

[0261]

[0262]

[0263] 2.2 Growth curves of three streptococcal strains and one new strain under the action of tandem antimicrobial peptide BA2

[0264] Due to limitations in experimental time and antimicrobial peptide content, the growth curve experiment only investigated the effect of BA2 antimicrobial peptide on the growth curves of three streptococcal strains and one new strain. The OD values ​​of the bacterial culture were measured at specific time points. 600 Numerical value, OD 600 The numerical values ​​reflect the bacterial concentration, and the colony count is calculated after 24 hours when 10 μL of bacterial solution is taken at a specific time, inoculated into solid GAM medium using a serial dilution method. The colony count reflects the number of viable bacteria at a specific time point.

[0265] Growth curves of *Streptococcus griseus* showed that GST protein slightly promoted bacterial growth. The bacterial culture containing GST protein entered the logarithmic growth phase at 4 hours, while the culture without GST protein entered the logarithmic growth phase at 5 hours. Furthermore, the final concentration of the culture containing GST protein was higher than that without GST protein, consistent with the 96-well plate inhibition experiment. 10 μg / mL Nisin did not inhibit the growth of *Streptococcus griseus*, and the strain entered the logarithmic growth phase at 5 hours, with a higher final concentration than that in the GAM-only anaerobic medium. 100 μg / mL BA2 showed no inhibitory effect on *Streptococcus griseus*, while 250 μg / mL BA2 showed excellent inhibitory effect, and the inhibitory effect of 250 μg / mL BA2 was better than that of 50 μg / mL Kan, consistent with the 96-well plate inhibition experiment. Figure 5 and Figure 6 As shown. Among them, Figure 5 The growth curves for different concentrations of BA2 and the control are shown, with the ordinate representing the OD value and the abscissa representing the culture time. Figure 6 The growth curves for 250 μg / mL BA2 and the control are shown, with CFU on the ordinate and culture time on the abscissa.

[0266] Growth curves of *Streptococcus sanguinis* showed that GST protein did not affect the growth of the strain. Both cultures containing and without GST protein entered the logarithmic growth phase at 4 hours, consistent with the 96-well plate inhibition assay. 10 μg / mL Nisin, 50 μg / mL Kan, and 250 μg / mL BA2 all inhibited the growth of *Streptococcus sanguinis*, again consistent with the 96-well plate inhibition assay. *Streptococcus sanguinis* entered the plateau phase at 10 hours, at which point the OD... 595The value is 0.6, and the number of viable bacteria in 10 μL of bacterial solution is 7 × 10⁶. 7 The growth curve of Streptococcus sanguinis is as follows: Figure 7 and Figure 8 As shown. Among them, Figure 7 The growth curves for different concentrations of BA2 and the control are shown, with the ordinate representing the OD value and the abscissa representing the culture time. Figure 8 The growth curves for 250 μg / mL BA2 and the control are shown, with CFU on the ordinate and culture time on the abscissa.

[0267] Growth curves of *Streptococcus mutans* showed that GST protein promoted its growth, with the final concentration consistent with that of the GAM-only anaerobic medium. 10 μg / mL Nisin did not inhibit *Streptococcus mutans* growth, and its growth curve coincided with that of the strain in the GAM-only anaerobic medium. 250 μg / mL BA2 showed slight inhibitory activity against *Streptococcus mutans*, leading to a decrease in the final bacterial concentration, but the inhibitory effect was poor. Among the five control groups, 50 μg / mL Kan showed the best inhibitory effect, but the inhibitory effect was not significant and did not change the final bacterial concentration. The OD595 value of *Streptococcus mutans* at 14 hours was 0.85, and the viable cell count in 10 μL of bacterial solution was 1.1 × 10⁻⁶. 7 Streptococcus mutans containing 250 μg / ml BA2 had an OD595 value of 0.7 at 14 hours, and the viable count in 10 μL of bacterial culture was 0.9 × 10⁻⁶. 7 The growth curve of Streptococcus mutans is as follows: Figure 9 and Figure 10 As shown. Among them, Figure 9 The growth curves for different concentrations of BA2 and the control are shown, with the ordinate representing the OD value and the abscissa representing the culture time. Figure 10 The growth curves for 250 μg / mL BA2 and the control are shown, with CFU on the ordinate and culture time on the abscissa.

[0268] The above description, in conjunction with specific embodiments, provides a detailed explanation of this application and should not be construed as limiting the implementation of this application to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the basic inventive concept of this application.

Claims

1. A salivary streptococcal antimicrobial peptide, characterized in that: The antimicrobial peptide from *Streptococcus saliva* was derived from the pGEX-6P-1-BB recombinant plasmid in *Escherichia coli* (…). Escherichia coli The pGEX-6P-1-BB recombinant plasmid was obtained by inducing expression in BL21(DE3) and then purifying via GST affinity. The plasmid was constructed as follows: a BamHI restriction site was added to the 5' end of the target nucleotide shown in SEQ ID NO.13, and an XhoI restriction site was added to the 3' end. The pGEX-6P-1 plasmid was then double-digested with BamHI and XhoI, and the target nucleotide with the added restriction sites was ligated to the double-digested pGEX-6P-1 plasmid. The GST tag in the *Streptococcus salivarius* antimicrobial peptide was provided by the pGEX-6P-1 expression vector. The *Streptococcus salivarius* antimicrobial peptide contained the GST tag and a BB tandem peptide encoded by the nucleotide sequence shown in SEQ ID NO.

13. The amino acid sequence of the BB tandem peptide was: GGGVIQTISHECRMNSWQFLFTCCSGSGGSGGGGVIQTISHECRMNSWQFLFTCCS.

2. The antimicrobial peptide for *Streptococcus salivans* as described in claim 1, used in the preparation of a peptide for in vitro inhibition of *Streptococcus griseus* (… Streptococcus gordonii ) and / or Streptococcus sanguinis ( Streptococcus sanguinis The application of the reagent, wherein the concentration of the salicylic streptococcal antimicrobial peptide is 200 ng / μL.

3. A method for preparing the *Streptococcus salivarius* antimicrobial peptide of claim 1, characterized in that, Includes the following steps: (1) Add a BamHI restriction site to the 5' end and an XhoI restriction site to the 3' end of the target nucleotide shown in SEQ ID NO.

13. Digest the pGEX-6P-1 plasmid with BamHI and XhoI, and use T4 DNA Ligase to ligate the target nucleotide with the added restriction sites to the double-digested pGEX-6P-1 plasmid to obtain the pGEX-6P-1-BB recombinant plasmid. (2) The pGEX-6P-1-BB recombinant plasmid was transformed into Escherichia coli DH5α for cloning, and the recombinant plasmid was identified by enzyme digestion and sequenced for confirmation. (3) The pGEX-6P-1-BB recombinant plasmid confirmed by sequencing was transformed into Escherichia coli BL21(DE3), and the expression of the salivary streptococcal antimicrobial peptide was induced by IPTG. (4) The cells were broken and the antimicrobial peptides of Streptococcus salivarius were isolated by GST affinity purification.

4. The method according to claim 3, characterized in that, In step (2), ScaI was used to digest and identify the pGEX-6P-1-BB recombinant plasmid.

5. The method according to claim 3 or 4, characterized in that, In step (3), the final IPTG concentration was 0.5 mM, the induction temperature was 15℃, and the induction time was 16 h.

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