A recombinant Bacillus displaying a Streptococcus suis prophage lysin, its construction method, and its application.

Abstract

This invention provides a recombinant Bacillus subtilis displaying a prephage lysin from Streptococcus suis, its construction method, and its applications, belonging to the field of genetic engineering technology. This invention fuses the cotG gene encoding the anchoring protein and the Lys0859 gene encoding the prephage lysin from Bacillus subtilis, and expresses them exogenously in Bacillus subtilis to obtain recombinant Bacillus subtilis displaying the prephage lysin Lys0859 on its surface. The spores produced by the recombinant Bacillus subtilis are resistant to high temperatures, strong acids and alkalis, and the gastrointestinal environment, and exhibit antibacterial effects against Gram-positive bacteria such as Streptococcus suis, Staphylococcus aureus, Listeria monocytogenes, and Clostridium perfringens, making them a novel antibiotic alternative. When applied to Streptococcus suis infections, the recombinant spores can be administered orally, offering significant convenience in storage and transportation, and also saving production costs.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a recombinant Bacillus displaying a Streptococcus suis prophage lysin, its construction method, and its application. Background Technology

[0002] With the full implementation of my country's "antibiotic ban" in feed, the feed industry has entered a new era of prohibiting antibiotic use, making the search for more, better, and more effective antibiotic alternatives an urgent priority. Phage lyases are cell wall hydrolases synthesized in the host during the later stages of double-stranded DNA bacteriophage infection. Through perforin, the lyase reaches the host cell's peptidoglycan layer to perform its lysis function, ultimately releasing progeny phages. Phage lyases have become a research hotspot due to their advantages such as low resistance, specificity, high efficiency, and specific bactericidal activity. The reported suicidal streptococcal prophage lyase Lys0859 possesses a CHAP catalytic domain (EAD) and an SH3b cell wall binding domain (CBD). This lyase is a cysteine- and histidine-dependent amide hydrolase. However, this lyase has extremely poor tolerance to the gastrointestinal environment, is unstable, and easily degrades, increasing storage and transportation costs. Injection administration causes significant stress to pigs, affecting piglet production performance and increasing workload and feeding costs due to operational inconvenience. On the other hand, the preparation process of phage lysins, such as purification and disruption, is costly. Therefore, there is an urgent need to find a method for preparing phage lysins. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a recombinant Bacillus strain exhibiting Streptococcus suis prophage lysin, which is not only resistant to high temperature, strong acid and alkali and gastrointestinal environment, but also has good specific bactericidal activity, providing a novel antibiotic alternative for the prevention of Streptococcus suis infection.

[0004] This invention provides a recombinant Bacillus strain displaying a prephage lysin from Streptococcus suis, wherein the prephage lysin Lys0859 is displayed on the surface of the Bacillus' anchoring protein.

[0005] Preferably, the Bacillus anchoring protein is cotG protein;

[0006] The amino acid sequence of the cotG protein is shown in SEQ ID NO: 1.

[0007] Preferably, the amino acid sequence of the prephage lysin Lys0859 is shown in SEQ ID NO: 2.

[0008] Preferably, the amino acid sequence of the fusion protein formed by the anchoring protein and the prephage lysin Lys0859 is shown in SEQ ID NO: 3.

[0009] Preferably, the host strain of the recombinant Bacillus is the Bacillus subtilis 168 standard strain.

[0010] This invention provides a method for constructing the recombinant Bacillus, comprising the following steps:

[0011] A fusion gene formed by Bacillus anchoring protein and prophage lysin Lys0859 was prepared.

[0012] The fusion gene was cloned into a shuttle plasmid to obtain a recombinant plasmid;

[0013] The recombinant plasmid was transformed into a Bacillus host bacterium to obtain recombinant Bacillus bacterium displaying a Streptococcus suis prophage lysin.

[0014] Preferably, the method for preparing the fusion gene formed by Bacillus anchoring protein and prophage lyase Lys0859 includes amplifying the coding gene of Bacillus anchoring protein and the coding gene of prophage lyase Lys0859 respectively, and obtaining the fusion gene cotG-0859 by overlapping PCR amplification.

[0015] Preferably, the primers for amplifying the encoding gene of Bacillus anchoring protein include a forward primer with a nucleotide sequence as shown in SEQ ID NO: 4 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 5;

[0016] Primers for amplifying the encoding gene of the prephage lysin Lys0859 include a forward primer with a nucleotide sequence as shown in SEQ ID NO: 6 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 7;

[0017] Primers used for overlap PCR amplification include a forward primer with a nucleotide sequence as shown in SEQ ID NO: 4 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 7.

[0018] Preferably, the fusion gene is integrated into the amylase gene in the host genome of Bacillus subtilis.

[0019] The present invention also provides recombinant spores produced by the aforementioned recombinant Bacillus.

[0020] The present invention provides the application of the recombinant Bacillus, the recombinant Bacillus obtained by the construction method, or the recombinant spores in the preparation of antimicrobial agents or feed, wherein the antimicrobial agent preferably includes at least one of the following: Streptococcus suis antimicrobial agent, Staphylococcus aureus antimicrobial agent, Listeria monocytogenes antimicrobial agent, and Clostridium perfringens antimicrobial agent.

[0021] This invention provides a recombinant Bacillus strain displaying a prephage lysin from Streptococcus suis, specifically displaying the prephage lysin Lys0859 on the surface of the Bacillus' anchoring protein. During endophageal spore formation, the Lys0859 gene is successfully displayed on the spore surface along with the expression of the anchoring protein. This invention uses SDS-PAGE, Western blot, and fluorescence immunoassay to confirm the successful immobilization of the prephage lysin on the spore surface. To verify whether the exogenous protein affects spore function, this invention measures the enzyme production, growth curves, and spore formation rate of wild-type and recombinant strains. The results show that the expression of the fusion protein does not affect the spore-forming function of the Bacillus. Furthermore, the stability of the recombinant spores was determined by treating them with high temperature, strong acids and alkalis, ethanol, artificial gastric fluid, artificial intestinal fluid, bile salts, metal ions, and EDTA. The results indicate that the recombinant spores have strong tolerance to these environments. Meanwhile, the recombinant Bacillus, due to the expression of prophage lysin on its surface, exhibits good antibacterial activity against Gram-positive bacteria such as Streptococcus suis, Staphylococcus aureus, Listeria monocytogenes, and Clostridium perfringens. Therefore, the recombinant spores produced by the recombinant Bacillus provided by this invention not only possess stable environmental adaptability but also maintain good specific bactericidal properties, providing an antibacterial agent for the development of prevention of Streptococcus suis infection. Furthermore, it has unique advantages in oral inoculation, storage, and transportation. Attached Figure Description

[0022] Figure 1 Electrophoresis diagrams of gene fragment amplification; (a) Electrophoresis diagram of cotG and 0859 gene amplification; (b) Electrophoresis diagram of fusion gene fragment cotG-0859 amplification; (c) Electrophoresis diagram of plasmid pDG364-cotG-0859 after double digestion with HindIII and EcoRI in lane 2; (d) PCR electrophoresis diagram of recombinant cotG-0859 / DH5α colony.

[0023] Figure 2 Recombinant Bacillus subtilis rBS coG-0859 Results of amylase activity assay;

[0024] Figure 3 Recombinant Bacillus subtilis rBS coG-0859 Results of bacterial culture PCR identification;

[0025] Figure 4 Recombinant Bacillus subtilis rBS cotG-0859 Genomic PCR identification results; M: Marker, W: strain 168, N2: rBS cotG-0859 ;

[0026] Figure 5 The antibacterial effect of recombinant Bacillus subtilis;

[0027] Figure 6 Wild-type Bacillus subtilis 168 and recombinant Bacillus subtilis rBS cotG-0859 The first-step growth curve;

[0028] Figure 7 (a) shows wild-type Bacillus subtilis 168 and recombinant Bacillus subtilis rBS. cotG-0859 A graph showing the total number of bacteria; Figure 7 (b) shows wild-type Bacillus subtilis 168 and recombinant Bacillus subtilis rBS. cotG-0859 A diagram of budding;

[0029] Figure 8 For SDS-PAGE analysis results, M: Marker 1: pCold-0859, 2: pCold-cotG-0859, 3: rBS cotG-0859 ;

[0030] Figure 9 Westem-blotting analysis results of anti-0859 polyclonal antibody: M: Marker, 1: rBScotG-0859, 2: pCold-cotG-0859, 3: pCold-0859, 4: strain 168;

[0031] Figure 10 Immunofluorescence analysis graph;

[0032] Figure 11 This invention provides a technical route for constructing recombinant Bacillus strains that exhibit Streptococcus suis prophage lysin. Detailed Implementation

[0033] This invention provides a recombinant Bacillus strain that displays a prephage lysin from Streptococcus suis, and expresses the prephage lysin Lys0859 on the surface of the Bacillus anchoring protein.

[0034] In this invention, the Bacillus anchoring protein preferably includes the cotG protein. The amino acid sequence of the cotG protein is preferably as shown in SEQ ID NO: 1 (MLGHYSHSDIEEAVKSAKKEGLKDYLYQEPHGKKRSHKKSHRTHKKSRSHKKSYCSHKKSRSHKKSFCSHKKSRSHKKSYCSHKKSRSHKKSYRSHKKSRSYKKSYRSYKKSRSYKKSCRSYKKSRSYKKSYCSHKKKSRSYKKSCRTHKKSYRSHKKYYKKPHHHCDDYKRHDDYDSKKEYWKDGNCWVVKKKYK). The preferred amino acid sequence of the prephage lysin Lys0859 is as shown in SEQ ID NO: 2(GGSSGSMTTVFEVVNFAKDLANRGQGVDYDGWYGNQCVDLPNWISGKFFGKALWGNAIDLIKSAKQHGFEVHYMPTSERPRPGAIFVKNYWANDGGNYGHTGLIIGVSGNTVQTIEQNLVGNLSVGGPAQYSSQQISNLVGWFYPPYSDSTAVATQSSSGNLGKVKDEQGTMTVKVSLLNVRDKPGLDGKVVATYTNSEQFNYDSVYIADGYIWVSYVSRSGVRRYVAAGEESNRRNVVPYGIFKHHHHHHH).In this embodiment of the invention, the fusion protein is the linking sequence of Bacillus anchoring protein-prophage lysin Lys0859, and the corresponding amino acid sequence is preferably as shown in SEQ ID NO: 3(MLGHYSHSDIEEAVKSAKKEGLKDYLYQEPHGKKRSHKKSHRTHKKSRSHKKSYCSHKKSRSHKKSFCSHKKSRSHKKSYCSHKKSRSHKKSYRSHKKSRSYKKSYRSYKKSRSYKKSCRSYKKSRSYKKSYCSHKKKSRSYKKSCRTHKKSYRSHKKYYKKPHHHCDDYKRHDDYDSKKEYWKDGNCWVVKKKYKGGSSGSMTTVFEVVNFAKDLANRGQGV) As shown in the diagram (DYDGWYGNQCVDLPNWISGKFFGKALWGNAIDLIKSAKQHGFEVHYMPTSERPRPGAIFVKNYWANDGGNYGHTGLIIGVSGNTVQTIEQNLVGNLSVGGPAQYSSQQISNLVGWFYPPYSDSTAVATQSSSGNLGKVKDEQGTMTVKVSLLNVRDKPGLDGKVVATYTNSEQFNYDSVYIADGYIWVSYVSRSGVRRYVAAGEESNRRNVVPYGIFKHHHHHHH), the prephage lysin Lys0859 was successfully displayed on the surface of recombinant Bacillus via an anchoring protein.

[0035] This invention does not impose any particular limitation on the type of host bacillus; any host bacillus well-known in the art can be used, such as Bacillus subtilis strain 168. The cotG protein is preferably derived from Bacillus subtilis strain 168.

[0036] This invention provides a method for constructing the recombinant Bacillus, comprising the following steps:

[0037] A fusion gene formed by Bacillus anchoring protein and prophage lysin Lys0859 was prepared.

[0038] The fusion gene was cloned into a shuttle plasmid to obtain a recombinant plasmid;

[0039] The recombinant plasmid was transformed into a Bacillus host bacterium to obtain recombinant Bacillus bacterium displaying a Streptococcus suis prophage lysin.

[0040] This invention first prepares a fusion gene formed by Bacillus anchoring protein and prophage lysin Lys0859.

[0041] In this invention, the method for preparing the fusion gene formed by Bacillus anchoring protein and prophage lyase Lys0859 preferably includes amplifying the coding gene of Bacillus anchoring protein and the coding gene of prophage lyase Lys0859 respectively, and obtaining the fusion gene cotG-0859 by overlapping PCR amplification.

[0042] In this invention, the primers for amplifying the encoding gene of Bacillus anchoring protein preferably include a forward primer with the nucleotide sequence shown in SEQ ID NO: 4 and a reverse primer with the nucleotide sequence shown in SEQ ID NO: 5. The preferred reaction program for amplifying the encoding gene of Bacillus anchoring protein is 98℃ for 10 min; 98℃ for 10 s, 55℃ for 5 s, 72℃ for 20 s, for a total of 35 cycles; 72℃ for 5 min. The primers for amplifying the encoding gene of prephage lysin Lys0859 preferably include a forward primer with the nucleotide sequence shown in SEQ ID NO: 6 and a reverse primer with the nucleotide sequence shown in SEQ ID NO: 7. The preferred reaction program for amplifying the encoding gene of prephage lysin Lys0859 is 98℃ for 10 min; 98℃ for 10 s, 55℃ for 5 s, 72℃ for 30 s, for a total of 35 cycles; 72℃ for 5 min.

[0043] In this invention, the primers for overlapping PCR amplification include a forward primer with a preferred nucleotide sequence as shown in SEQ ID NO: 4 and a reverse primer with a preferred nucleotide sequence as shown in SEQ ID NO: 7, wherein the forward primer shown in SEQ ID NO: 4 has a HindIII restriction site and the reverse primer shown in SEQ ID NO: 7 has an EcoRI restriction site.

[0044] After obtaining the fusion gene, the present invention clones the fusion gene into a shuttle plasmid to obtain a recombinant plasmid.

[0045] This invention does not impose any particular limitation on the type of shuttle plasmid; any shuttle plasmid well-known in the art, such as the pDG364 plasmid, can be used. The cloning method preferably includes homologous recombination, specifically involving double digestion of the shuttle plasmid and the fusion fragment cotG-0859 with restriction endonucleases HindIII and EcoRI. The digestion products are then purified and ligated to verify and identify positive recombinant plasmids.

[0046] The present invention transforms the recombinant plasmid into a Bacillus host bacterium to obtain recombinant Bacillus bacterium displaying a Streptococcus suis prophage lysin.

[0047] In this invention, the preferred transformation method involves linearizing the recombinant plasmid and then chemically transforming it into competent cells of the Bacillus subtilis host bacteria. The fusion gene is preferably integrated into the amylase gene in the genome of the Bacillus subtilis host bacteria. The preferred integration method involves cloning the upper and lower homologous arms of the amyE (amylase) gene into the Escherichia coli-Bacillus subtilis shuttle plasmid pDG364, introducing the recombinant plasmid pDG364-cotG-0859 into competent Bacillus subtilis cells, and then integrating the exogenous fusion fragment cotG-0859 into the amyE gene of Bacillus subtilis through a double crossover of homologous arms.

[0048] In this invention, the identification of recombinant Bacillus strains exhibiting the lysinus prophage lysin is preferably included. The identification method preferably includes amylase activity identification, bacterial culture PCR identification, and genome identification. Since the fusion gene is preferably integrated into the amylase gene in the host Bacillus subtilis genome, affecting amylase activity, strains lacking amylase activity are selected for bacterial culture PCR identification. The primers used for the bacterial culture PCR identification include amyE-F (SEQ ID NO: 10) / amyE-R (SEQ ID NO: 11) and BS168-F (SEQ ID NO: 12) / BS168-R (SEQ ID NO: 13). The primers used for genome identification included amyE-F (SEQ ID NO: 10) / amyE-R (SEQ ID NO: 11), G8-F (SEQ ID NO: 8) / G8-R (SEQ ID NO: 9), amyE-F (SEQ ID NO: 10) / G8-R (SEQ ID NO: 9), and G8-F (SEQ ID NO: 8) / amyE-R (SEQ ID NO: 11). All three identification results showed that the present invention successfully inserted cotG-0859 into the amylase gene of Bacillus subtilis, obtaining a recombinant Bacillus exhibiting the lysinus prophage lysinus prostreptococcus.

[0049] The present invention also provides a recombinant spore produced by the recombinant Bacillus.

[0050] In this invention, the preferred method for preparing the recombinant spores is to inoculate activated recombinant Bacillus subtilis into DSM medium, culture at 37°C and 180 rpm for 48 h, collect the bacterial cells, wash them, and obtain recombinant Bacillus subtilis spores. The inoculation amount of the activated recombinant Bacillus subtilis is preferably 0.8–1.2%, more preferably 1.0%. The method for collecting the bacterial cells preferably includes centrifugation, with a centrifugation speed preferably of 8500 rpm, more preferably 10 min. The washing solution preferably includes PBS. The recombinant Bacillus spores also preferably include purification. The purification method preferably involves treating with lysozyme, collecting the precipitate, washing with sodium chloride solution, resuspending the precipitate in Tris-HCl containing lysozyme, treating at 37°C for 1 h, centrifuging again to collect the precipitate, and then washing with 1 mol / L NaCl deionized water, 0.05% SDS, and TEP buffer to obtain pure spores.

[0051] In this invention, the recombinant Bacillus spores exhibit good temperature tolerance, pH tolerance, tolerance to artificial gastric juice, tolerance to bile salts, tolerance to artificial intestinal juice, and tolerance to metal ions and EDTA. Furthermore, the recombinant Bacillus spores show varying degrees of antibacterial effects against different serotypes of Streptococcus suis, Staphylococcus aureus, Listeria monocytogenes, and Clostridium perfringens, while wild-type Bacillus subtilis only shows antibacterial effects against Staphylococcus aureus.

[0052] The present invention provides the application of the recombinant Bacillus, the recombinant Bacillus obtained by the construction method, or the recombinant spores in the preparation of antimicrobial agents or feed, wherein the antimicrobial agent preferably includes at least one of the following: Streptococcus suis antimicrobial agent, Staphylococcus aureus antimicrobial agent, Listeria monocytogenes antimicrobial agent, and Clostridium perfringens antimicrobial agent.

[0053] The following detailed description, in conjunction with embodiments, illustrates a recombinant Bacillus sp. displaying a Streptococcus suis prophage lysin, its construction method, and its applications. However, these descriptions should not be construed as limiting the scope of protection of this invention.

[0054] The following examples are used to illustrate the present invention, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in this study are all commonly used reagents.

[0055] Example 1

[0056] Construction method of recombinant shuttle plasmid pDG364-cotG-0859

[0057] 1. Reagent preparation

[0058] ①50×TAE formula: Weigh 242g Tris, 37.2g Na2EDTA, measure 57.1mL glacial acetic acid, and add deionized water to make up to 1L.

[0059] ②1% agarose gel: Dissolve 1g agarose in 100mL 1×TAE, heat in a microwave oven to mix evenly, add 3μL nucleic acid dye and mix evenly, pour into a horizontal gel frame, insert a comb and let it cool naturally.

[0060] 2. PCR amplification of the anchoring protein cotG

[0061] Using the genome of wild-type Bacillus subtilis 168 (purchased from the China Center for Type Culture Collection) as a template, and cotG-F and cotG-R as primers, the cotG target fragment was amplified by PCR. The PCR amplification system is shown in Table 1, and the PCR amplification procedure is shown in Table 2. The obtained PCR products were examined by 1% agarose gel electrophoresis. The theoretical size of the cotG fragment is 613 bp. Figure 1 In section (a), after verification, the gel was recovered using the San Prep column DNA gel recovery kit and stored at -20°C for later use.

[0062] Table 1. PCR amplification system for cotG fragment

[0063]

[0064] Table 2 PCR amplification program for the cotG fragment

[0065]

[0066] Upstream primer cotG-F: 5′-CG AAGCTT ATGTTGGGCCACTATTCCCATTCTGACATCGAAG-3′(SEQ ID NO: 4), where the underlined part is the HindIII restriction site.

[0067] The downstream primer cotG-R: 5′-TCGATCCAGACGAGCCTCCTTTGTATTTCTTTTTGACTACCCAGCAATTG-3′ (SEQ ID NO: 5). The nucleotide sequence of the amplified product is shown in SEQ ID NO: 14().

[0068] 3. PCR amplification of the prophage lysin Lys0859

[0069] Using the genome of *Streptococcus suis* 0859 as a template, and primers 0859-F and 0859-R, the target fragment of the prephage lysin Lys0859 was amplified by PCR. The PCR amplification system is shown in Table 3, and the PCR amplification program is shown in Table 4. The PCR products were examined using 1% agarose gel electrophoresis. The theoretical size of the 0859 fragment is 764 bp. Figure 1 In section (a), after verification, the gel was recovered using the San Prep column DNA gel recovery kit and stored at -20°C for later use.

[0070] Table 3 PCR amplification system for fragment 0859

[0071]

[0072] Table 4. PCR amplification program for fragment 0859

[0073]

[0074] Upstream primer 0859-F: 5′-AAGGAGGCTCGTCTGGATCGATGACAACAGTATTTGAAGTAGTCAATTTTG-3′ (SEQ ID NO: 6);

[0075] Downstream primer 0859-R: 5′-CG GAATTCTTAGTGGTGGTGGTGGTGGTGTTTGAAAATACCATAAGGCACA-3′ (SEQ ID NO: 7), the underlined part is the EcoRI restriction site). The nucleotide sequence of the amplified product is as shown in SEQ ID NO: 15().

[0076] 4. Fusion of anchoring protein cotG and phage lysin Lys0859

[0077] The fusion gene fragment cotG-0859 was obtained using overlap PCR. The PCR amplification system is shown in Table 5, and the PCR amplification procedure is shown in Table 6. The PCR products were verified by 1% agarose gel electrophoresis. The theoretical size of the cotG-0859 fragment is 1356 bp. Figure 1 In section (b), after verification, the gel was recovered using the San Prep column DNA gel recovery kit and stored at -20°C for later use.

[0078] Table 5. PCR amplification system for the cotG-0859 fragment.

[0079]

[0080] Table 6 PCR amplification program for the cotG-0859 fragment

[0081]

[0082] Upstream primer cotG-F: 5′-CG AAGCTT ATGTTGGGCCACTATTCCCATTCTGACATCGAAG-3168 (SEQ ID NO: 4, underlined is the HindIII restriction site)

[0083] Downstream primer 0859-R: 5′-CG GAATTC

[0084] 5. Enzyme digestion of the fusion gene fragment cotG-0859 and the pDG364 vector

[0085] The pDG364 plasmid containing the upper and lower homologous arms of the amyE (amylase) gene and the fusion fragment cotG-0859 were double-digested using the restriction endonucleases HindIII and EcoRI. The digestion system for the cotG-0859 fusion fragment is shown in Table 7, and the digestion system for the pDG364 plasmid is shown in Table 8. Digestion conditions: 37℃ water bath for 1 h. The digestion products were verified by 1% agarose gel electrophoresis. After verification, the DNA was recovered using a San Prep column DNA gel extraction kit and stored at -20℃ for later use.

[0086] Table 7 Enzyme digestion system of fusion fragment cotG-0859

[0087]

[0088] Table 8. pDG364 plasmid digestion system

[0089]

[0090] 6. Connection

[0091] The digested fusion gene cotG-0859 and the pDG364 plasmid were ligated using T4 DNA ligase. The ligation conditions were 4℃ for 12 h. The ligation system is shown in Table 9.

[0092] Table 9 Connection System

[0093]

[0094] 7. Transformation

[0095] 1) Culture medium preparation: ① LB medium formulation: 10g peptone, 5g yeast extract, 10g NaCl, bring to a final volume of 1L with deionized water, pH 7.0, autoclave at 121℃ for 20min. ② 100mg / mL ampicillin formulation: Dissolve 1g ampicillin in 10mL of water, mix thoroughly, and sterilize using a 0.22μm filter. ③ LA plate formulation for ampicillin (final concentration 100μg / mL): 10g peptone, 5g yeast extract, 10g NaCl, 14g agar, bring to a final volume of 1L with deionized water, pH 7.0, autoclave at 121℃ for 20min. After cooling to 55℃, add 1mL of 100mg / mL ampicillin, mix thoroughly, pour into sterile petri dishes, and allow to solidify.

[0096] 2) Transformation

[0097] Thaw E. coli DH5α competent cells slowly on ice, and slowly add 20 μL of ligation product. Mix gently, incubate on ice for 30 min, then heat shock at 42°C for 90 s, and quickly cool on ice for 5 min. In a clean bench, add 900 μL of LB medium to EP tubes, mix thoroughly, and incubate at 37°C with shaking at 180 rpm for 1 h. Centrifuge at 5000 rpm for 5 min, remove some supernatant, and finally spread the bacterial culture evenly on LA plates containing ampicillin (final concentration 100 μg / mL) and incubate upside down in a 37°C incubator.

[0098] 8. Identification of positive clones

[0099] 1) Activation of the strain: After single bacteria have grown on the culture dish, pick the transformants. Inoculate the transformants into a bacterial bottle containing 5 mL LB (final concentration of ampicillin 100 μg / mL) and incubate at 37℃ with shaking at 180 rpm for 12-16 h.

[0100] 2) Bacterial culture PCR identification: A single colony of bacterial culture was used as a template, and G8-F / G8-R primers were used. The PCR reaction system is shown in Table 10. The PCR amplification procedure is shown in Table 11. The above PCR products were verified by 1% agarose gel electrophoresis, and their theoretical size of 1356 bp was confirmed to be correct. Figure 1 (c)

[0101] Table 10 PCR amplification system for transformant identification

[0102]

[0103]

[0104] Table 11 PCR amplification procedure for transformant identification

[0105]

[0106] Upstream primer G8-F: 5′-TTACGCGAAATACGGGCAGACAT-3′ (SEQ ID NO: 8);

[0107] Downstream primer G8-R: 5′-TTTTTAAAGGATTTGAGCGTAGCG-3′, SEQ ID NO: 9).

[0108] 3) Enzyme digestion identification: The positive clones were expanded and cultured. Recombinant plasmid pDG364-cotG-0859 was extracted using a plasmid extraction kit (Omega EZNAPlasmid Mini Kit I). The extracted recombinant plasmid was then subjected to enzyme digestion verification. The enzyme digestion system is shown in Table 12. Digestion conditions: 37℃ water bath for 1 h. The digestion products were examined using 1% agarose gel electrophoresis. The target band size was between 1000-2000, consistent with the target size. (See Table 12 for details). Figure 1 (d) The validated recombinant plasmid was sent to Qingke Biotechnology (Wuhan) Co., Ltd. for sequencing. The sequencing results were consistent with the original sequence, indicating that the recombinant plasmid pDG364-cotG-0859 was successfully constructed.

[0109] Table 12 Enzyme digestion system of recombinant plasmid pDG364-cotG-0859

[0110]

[0111] Example 2

[0112] Recombinant Bacillus subtilis rBS cotG-0859 Construction method

[0113] 1. Linearization of plasmids

[0114] The restriction enzyme digestion system for plasmid pDG364-cotG-0859 is shown in Table 13. The mixture was incubated at 37℃ for 1 hour. The results were verified by 1% agarose gel electrophoresis. After confirmation, the DNA was recovered using a San Prep column-based DNA gel extraction kit and stored at -20℃ for later use.

[0115] Table 13 Enzyme digestion system of plasmid pDG364-cotG-0859

[0116]

[0117] 2. Preparation of B. subitilis 168 competent cells

[0118] 1) Preparation of culture media: ① Preparation of 10×Spizizen's medium: 2g (NH4)2SO4, 14g K2HPO4, 6g KH2PO4, 1g sodium citrate, 100mL deionized water, autoclave at 121℃ for 20min, add 0.1ml 1mol / L MgSO4. ② Formulation of HS medium: Based on 10×Spizizen's medium, add 0.5% glucose, 50μg / mL DL-tryptophan, 50μg / mL uracil, 0.02% casein hydrolysate, 0.1% yeast extract, 8μg / mL arginine, 0.4μg / mL histidine, 1mmol / L MgSO4. ③ LS medium formulation: Based on 10×Spizizen's medium, add 0.5% glucose, 5 μg / mL DL-tryptophan, 5 μg / mL uracil, 0.01% casein hydrolysate, 0.1% yeast extract, 1 mmol / L MgSO4, 2.5 mM MgCl2, and 0.5 mM CaCl2. ④ 0.5 mol / L EGTA formulation: Weigh 19.02 g EGTA and dissolve it in 90 mL of deionized water. While stirring continuously, add 5 mol / L sodium hydroxide solution dropwise to maintain the pH at 8.0. Cool and transfer to a 100 mL volumetric flask to dilute to the mark.

[0119] 2) Preparation of B. subitilis 168 competent cells: B. subitilis 168 strain frozen in glycerol was streaked on LA plates and incubated at 37℃ for 12 h until single colonies appeared. Fresh B. subitilis 168 single colonies were inoculated into 5 mL of HS medium and incubated overnight at 37℃ in a shaker; 1 mL of overnight HS medium was inoculated into 20 mL of LS medium and incubated at 30℃ in a shaker for 3-4 h.

[0120] 3. The recombinant plasmid was transformed into B. subitilis 168 competent cells.

[0121] Take 1 mL of the above LS medium, add 20 μL of 0.5 mol / L EGTA, mix gently, and incubate at room temperature for 5 min; add 12 μL of linearized plasmid pDG364-cotG-0859, mix gently, and incubate at 37℃ and 180 rpm for 2 h; spread on chloramphenicol plates (final concentration 5 μg / mL) and incubate overnight at 37℃.

[0122] Example 3

[0123] Recombinant Bacillus subtilis rBS cotG-0859 Identification methods

[0124] 1. Identification of amylase activity in recombinant Bacillus subtilis

[0125] 1) Preparation of culture medium: ① Formula for LA medium containing starch: Add 1% soluble starch to LA medium, autoclave at 121℃ for 20 min, and after cooling to 55℃, pour into sterile petri dishes until solidified. ② 2mol / L NaCl: Weigh 11.6g of NaCl and dissolve it in 100mL of deionized water, mix well.

[0126] 2) Activation of Bacillus subtilis: Take recombinant Bacillus subtilis rBS frozen in glycerol cotG-0859 Strains of strain 1 and B. subtilis 168 were streaked on LA solid medium and incubated at 37°C for 12 h until single colonies appeared.

[0127] 3) Identification of amylase activity in Bacillus subtilis: Recombinant Bacillus subtilis rBS was picked up with a sterile pipette tip. cotG -0859 Single colonies of B. subtilis strain 168 were spotted on LA medium containing 1% starch. Three replicates were made for each strain, and the cultures were incubated at 37°C for 12 hours.

[0128] 4) Results of amylase production assay by the strain: The plates cultured for 12 h were stained with 5% iodine solution (purchased from Solarbio) for 5 min, and then washed twice with 2 mol / L NaCl. The results are shown in the figure. Figure 2 .

[0129] 2. PCR identification of recombinant Bacillus subtilis in bacterial culture

[0130] 1) Resuscitation of the strain: Recombinant Bacillus subtilis rBS frozen in glycerol was retrieved. cotG-0859 The strain was streaked on a chloramphenicol-resistant plate (5 μg / mL) and incubated at 37°C for 12 h until single colonies appeared. Single colonies from the chloramphenicol-resistant plate were picked and incubated on LB medium (5 μg / mL chloramphenicol) at 37°C for 18 h.

[0131] 2) PCR identification of the strain: The above recombinant Bacillus subtilis rBS cotG-0859 Using the bacterial strain as a template, PCR amplification was performed using amyE-F / amyE-R and BS168-F / BS168-R primers. The PCR products were further verified using 1% agar gel electrophoresis.

[0132] Upstream primer amyE-F: 5′-GGGATTTTTGACTCCGAAGTAAGTC-3′ (SEQ ID NO: 10);

[0133] Downstream primer amyE-R: 5′-ATTTGAGTTTATCACCCTTGTCACT-3′ (SEQ ID NO: 11);

[0134] Upstream primer BS168-F: 5′-CATTGATTTGTATTCACTCTGCCAAGTTG-3′ (SEQ ID NO: 12);

[0135] Downstream primer BS168-R: 5′-CATCAATGACCACAAGCTCATCTGTGAT-3′ (SEQ ID NO: 13).

[0136] 3) PCR identification results of bacterial culture: Recombinant Bacillus subtilis rBS cotG-0859 Using the strain as a template, PCR amplification was performed with amyE-F / amyE-R primers, yielding a theoretical fragment size of 4448 bp. Using BS168-F / BS168-R primers, the theoretical fragment size was 4800 bp. (See [link to relevant documentation]). Figure 3 .

[0137] 3. Genome identification of recombinant Bacillus subtilis

[0138] 1) Resuscitation of the strain: Recombinant Bacillus subtilis rBS frozen in glycerol was retrieved. cotG-0859 The strain and B. subtilis168 were streaked on LA solid medium and incubated at 37°C for 12 h until single colonies appeared. Single colonies were picked and transferred to 5 mL of LB medium and incubated at 37°C with shaking at 180 rpm for 8 h.

[0139] 2) Genomic extraction: Extract recombinant bacteria rBS according to the kit instructions (Kangwei Century). cotG-0859 The genome of wild fungus B. subtilis 168.

[0140] 3) Genomic PCR identification: Using the extracted genome as a template, PCR amplification was performed using amyE-F / amyE-R, G8-F / G8-R, amyE-F / G8-R, and G8-F / amyE-R as primers. The PCR reaction system is shown in Table 14. The PCR amplification program is shown in Table 15.

[0141] Table 14 Genomic PCR Amplification System

[0142]

[0143] Table 15 Genomic identification PCR amplification procedure

[0144]

[0145] Upstream primer G8-F: 5′-TTACGCGAAATACGGGCAGACAT-3′ (SEQ ID NO: 8);

[0146] Downstream primer G8-R: 5′-TTTTTAAAGGATTTGAGCGTAGCG-3′, SEQ ID NO: 9).

[0147] Upstream primer amyE-F: 5′-GGGATTTTTGACTCCGAAGTAAGTC-3′ (SEQ ID NO: 10);

[0148] Downstream primer amyE-R: 5′-ATTTGAGTTTATCACCCTTGTCACT-3′ (SEQ ID NO: 11);

[0149] 3) Genomic PCR identification results: PCR products were further verified using 1% agar gel electrophoresis. Using primers amyE-F / amyE-R, wild-type and recombinant bacteria amplified to 2000 bp and 4448 bp, respectively; primers G8-F / G8-R amplified 0 bp and 1544 bp, respectively; primers amyE-F / G8-R amplified 0 bp and 2161 bp, respectively; and primers G8-F / amyE-R amplified 0 bp and 3562 bp, respectively. Except for amyE-F / amyE-R, no other primers showed specific bands in wild-type Bacillus subtilis, indicating that the gene fragment Em... r -cotG-0859 was successfully inserted into the amylase gene of Bacillus subtilis. (See attached image) Figure 4 .

[0150] Example 4

[0151] Preparation method of Bacillus subtilis spores

[0152] 1. Preparation of DSM medium: 0.8% nutrient broth, 0.1% KCl, 0.025% MgSO4·7H2O, 0.01 mmol MnCl2, adjust pH to 7.2, autoclave at 121℃ for 20 min, cool the medium to room temperature, add 1.0 μmol / L FeSO4 (prepared fresh for use) and 1.0 mmol / L CaCl2 (prepared fresh for use) after filtration sterilization.

[0153] 2. Reagent preparation: ① 30 mL 0.5 mol / L NaCl: Weigh 0.87 g NaCl and dissolve in 30 mL deionized water, mix well; ② 30 mL Tris-HCl (50 mmol / L, pH 7.2; containing lysozyme at a final concentration of 50 μg / mL): Weigh 0.2355 g Tris-HCl and dissolve in 30 mL deionized water, then add 1.5 mg lysozyme and mix well; ③ 1 mol / L NaCl: Dissolve 1.74 g NaCl in 30 mL deionized water and mix well; ④ 0.05% SDS: Dissolve 0.05 g SDS in 100 mL deionized water and mix well; ⑤ TEP buffer preparation: First, prepare 200 mM PMSF: Dissolve 0.3484 g PMSF in 10 mL isopropanol and mix well. Dissolve 0.2355g Tris-HCl in 30mL of deionized water, then add 0.1116g EDTA, and finally add 300μL of 200mM PMSF and mix well.

[0154] 3. Resuscitation of the strain: Same as step 3 in Example 3.

[0155] 4. Spore preparation: The activated strain was inoculated into DSM medium at a rate of 1% and cultured at 37℃ and 180 rpm for 48 h. The bacterial cells were collected by centrifugation at 8500 rpm for 10 min, and washed twice with PBS to obtain the spores of recombinant Bacillus subtilis.

[0156] 4. Purification of spores: Spores were purified using the lysozyme treatment method. The precipitate was washed with the following solutions, centrifuged at 8500 rpm for 10 min, and the supernatant was discarded and the precipitate was collected. (1) Wash the precipitate with 30 mL of 0.5 mol / L NaCl solution. (2) Resuspend the precipitate in 30 mL of Tris-HCl (50 mmol / L, pH 7.2; with a final lysozyme concentration of 50 μg / mL), treat at 37℃ for 1 h, centrifuge at 8500 rpm for 10 min, discard the supernatant and collect the precipitate. (3) 1 mol / L NaCl (4) Deionized water (5) 0.05% SDS (6) TEP buffer (7) Wash three times with deionized water. Finally, the spores were resuspended in sterile water and incubated at 65℃ for 1 h to kill non-spore-forming vegetative cells and obtain pure spores, which were stored at -20℃ for later use.

[0157] Example 5

[0158] Tolerance test of recombinant Bacillus subtilis spores

[0159] 1. Study on the temperature tolerance of recombinant Bacillus subtilis spores

[0160] 1) Take the recombinant spores rBS purified in Example 4 cotG-0859Treat at 65, 75, and 85℃ for 60 min. Samples were taken at 5, 10, 15, 30, and 60 min of incubation. The treated spores were then analyzed with sterile PBS for 10 minutes. -1 10 -2 10 -3 ...10-fold serial dilution; simultaneously, untreated spores were used as a control, and spore counts were performed. Finally, the spore survival rate was calculated. All samples were repeated 3 times, and the average was used as the standard.

[0161] 2) Recombinant spores rBS cotG-0859 Temperature tolerance test results: As shown in Table 16, the survival rate of recombinant spores decreased with the extension of time. The survival rate of recombinant spores treated at 65℃ for 60 min reached 91%, and the survival rate of recombinant spores treated at 75℃ for 60 min reached 85%. The survival rate of spores treated at 85℃ decreased significantly. These results indicate that recombinant spores have a moderate degree of resistance to temperature.

[0162] Table 16 Survival rate of recombinant Bacillus subtilis spores at different temperatures

[0163]

[0164] 2. Study on pH tolerance of recombinant Bacillus subtilis spores

[0165] 1) Preparation of different pH buffer solutions: Add dilute hydrochloric acid or sodium hydroxide to 0.85% physiological saline to adjust the pH to 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, and autoclave at 121℃ for 15 min.

[0166] 2) Take the recombinant spores rBS purified in Example 4 cotG-0859 The precipitate was inoculated in different pH buffer solutions and treated at 37°C for 30 min before sampling. Untreated spores were taken as a control. The spores were diluted, plated, and counted. Finally, the spore survival rate was calculated.

[0167] 3) Recombinant spore rBS cotG-0859 pH tolerance test results: As shown in Table 17, recombinant spore rBS cotG-0859 After 30 minutes of treatment, the survival rate of spores reached 70.76% in an acidic environment with pH=1, and the highest survival rate was observed at pH=7. In slightly acidic environments with pH=5 and 6, the survival rate of spores reached over 90%. Overall, spores are more stable in neutral environments and have strong resistance to strongly acidic environments.

[0168] Table 17 Survival rate of recombinant Bacillus subtilis spores under different pH conditions

[0169]

[0170] 3. Study on the tolerance of recombinant Bacillus subtilis spores to artificial gastric juice

[0171] 1) Preparation of artificial gastric fluid: Take 16.4 mL of concentrated hydrochloric acid and dilute it with physiological saline to make the pH 2.0. Then add 1% pepsin, stir thoroughly to dissolve, and filter with a 0.22 μm filter to remove bacteria.

[0172] 2) Take the recombinant spores rBS purified in Example 4 cotG-0859 The precipitate was inoculated into artificial gastric fluid and treated at 37°C for 60 min. Samples were taken at 5, 10, 15, 30, and 60 min of culture, with untreated spores taken as a control. The spores were diluted, plated, and counted. Finally, the spore survival rate was calculated.

[0173] 3) Recombinant spore rBS cotG-0859 Results of artificial gastric fluid tolerance test: As shown in Table 18, the survival rate of recombinant Bacillus subtilis spores gradually decreased with the extension of treatment time. The survival rate of recombinant spores reached 93% after 10 min of artificial gastric fluid treatment, and remained at 76% after 30 min and 1 h of treatment, indicating that recombinant Bacillus subtilis spores have strong tolerance to artificial gastric fluid.

[0174] Table 18 Survival rate of recombinant Bacillus subtilis spores in simulated gastric fluid

[0175]

[0176] 4. Study on bile salt tolerance of recombinant Bacillus subtilis spores

[0177] 1) Preparation of bile salts: Add porcine bile salts to physiological saline to make a mass fraction of 0.3%, mix thoroughly, and filter with a 0.22μm filter to remove bacteria.

[0178] 2) Recombinant spores treated with artificial gastric fluid for 1 hour were washed twice with sterile physiological saline. The spore precipitate was inoculated into 0.3% bile salt solution and treated at 37°C for 5 hours. Samples were taken at 1, 3, and 5 hours of culture, and untreated spores were taken as a control. The spores were diluted, plated, and counted. Finally, the spore survival rate was calculated.

[0179] 3) Recombinant spore rBS cotG-0859 Results of artificial bile salt tolerance test: As shown in Table 19, the survival rates of recombinant Bacillus subtilis spores after incubation in 0.3% bile salt at 37℃ for 1, 3 and 5 hours were 87.46%, 96.02% and 91.60%, respectively, indicating that the recombinant spores have strong tolerance to 0.3% porcine bile salt.

[0180] Table 19 Survival rate of recombinant Bacillus subtilis spores in 0.3% bile salts

[0181]

[0182] 5. Study on the tolerance of recombinant Bacillus subtilis spores to artificial intestinal fluid

[0183] 1) Preparation of artificial intestinal fluid: Weigh 6.8g of KH2PO4 and dissolve it in 800mL of deionized water. Adjust the pH to 6.8 with 0.1mol / L NaOH and bring the volume to 1L. Then add 1% trypsin and stir thoroughly until completely dissolved. Filter the solution through a 0.22μm filter to remove bacteria.

[0184] 2) Recombinant spores treated with artificial gastric fluid for 1 hour were washed twice with sterile physiological saline. The spore precipitate was inoculated into artificial intestinal fluid and treated at 37°C for 6 hours. Samples were taken at 0.5, 1, 2, 4, and 6 hours of culture, and untreated spores were taken as a control. The spores were diluted and plated for counting. Finally, the spore survival rate was calculated.

[0185] 3) Recombinant spore rBS cotG-0859 Results of artificial intestinal fluid tolerance test: As shown in Table 20, the survival rates of recombinant Bacillus subtilis after treatment in artificial intestinal fluid for 0.5h, 1h, 2h, 4h and 6h were 96.91%, 93.83%, 93.65%, 88.14% and 88.14%, respectively. The survival rate of spores did not change after 4h and 6h of treatment, indicating that the spores have a strong tolerance to artificial intestinal fluid.

[0186] Table 20 Survival rate of recombinant Bacillus subtilis spores in artificial intestinal fluid

[0187]

[0188]

[0189] 6. Study on the tolerance of recombinant Bacillus subtilis spores to metal ions and EDTA

[0190] 1) Preparation of metal ions and EDTA: Prepare 0.1 mol / L Cu 2+ 0.1 mol / L Ca 2+ 0.1 mol / L Mg 2+ 0.1 mol / L Mn 2+ 0.1 mol / L Zn 2+ 0.1 mol / L Ba 2+ 0.1 mol / L Fe 2+ 0.1 mol / L EDTA was filtered through a 0.22 μm filter for sterilization.

[0191] 2) Take the recombinant spores rBS purified in Example 4 cotG-0859The precipitate was inoculated in 0.1 mol / L metal ions and EDTA and treated at 37℃ for 1 h. Untreated spores were used as a control. The spores were diluted and plated for counting. Finally, the spore survival rate was calculated.

[0192] 3) Recombinant spore rBS cotG-0859 Results of the tolerance test to metal ions and EDTA: As shown in Table 21, the recombinant spores, after being treated with 0.1 mol / L metal ions and EDTA for 1 hour, achieved a survival rate of over 80%, and their Ca2+ tolerance was significantly lower. 2+ Ba 2+ The survival rate was about 75%, indicating that the recombinant spores had a strong tolerance to metal ions and EDTA.

[0193] Table 21 Survival rate of recombinant Bacillus subtilis spores in metal ions and EDTA.

[0194]

[0195] Example 6

[0196] Study on the antibacterial function of recombinant Bacillus subtilis spores

[0197] 1. Preparation of culture media: ① Preparation of TSA+5% FBS medium: Weigh 40g TSA and dissolve it in 1L deionized water, mix well, autoclave at 121℃ for 20min, and after cooling to 55℃, add 50mL calf serum and mix well. Pour into sterile petri dishes and let it solidify. ② Preparation of TSB medium: Weigh 30g TSB and dissolve it in 1L deionized water, mix well, and autoclave at 121℃ for 20min. ③ Preparation of semi-solid medium: Dissolve 2.5g TSA medium and 1.5g TSB in 100mL deionized water, mix well, and autoclave at 121℃ for 20min.

[0198] 2. Preparation of indicator bacteria: Different serotypes of Streptococcus suis and Listeria monocytogenes preserved in glycerol were streaked on TSA + 5% FBS solid medium and incubated at 37℃ for 12 h. Single colonies were picked and incubated overnight at 37℃ and 180 rpm in 5 mL TSB + 5% FBS liquid medium. The overnight culture was inoculated into 5 mL TSB + 5% FBS liquid medium and incubated at 37℃ and 180 rpm for 4 h to reach the logarithmic phase. The concentration was adjusted to 0.5 MCF using a McFarland turbidimeter.

[0199] 3. The Oxford cup method was used to verify the antibacterial effect: Using sterile forceps, pre-sterilized Oxford cups were gently and evenly placed into 90cm petri dishes containing TSA + 5% FBS. Six Oxford cups (recombinant spores rBS) were placed in each dish. cotG-0859Three replicates of *Bacillus subtilis* 168 and three replicates of wild-type *Bacillus subtilis* were prepared, ensuring a center-to-center distance of at least 24 mm and a distance of at least 15 mm between the Oxford cup and the edge of the culture dish. 0.1 mL of indicator bacterial suspension was mixed thoroughly with semi-solid culture medium at approximately 50°C and gently poured into the culture dish containing the Oxford cup, covering the entire medium. After the semi-solid medium solidified, the Oxford cup was removed, and 0.1 mL of purified recombinant spores was added to each. *B. subtilis* 168 was used as a control. The culture medium was labeled sequentially, and the dishes were gently incubated at 37°C for 12 hours. Three replicates were performed, and the average value was taken.

[0200] 4. Antibacterial Results of Recombinant Spores: The antibacterial diameters of the spores are shown in Table 22. Recombinant spores exhibited varying antibacterial effects against different serotypes of *Streptococcus suis*, *Staphylococcus aureus*, *Listeria monocytogenes*, and *Clostridium perfringens*. Wild-type *Bacillus subtilis* showed antibacterial activity against *Staphylococcus aureus*. The antibacterial results are shown in Table 22. Figure 5 .

[0201] Table 22 Inhibition diameters of wild-type and recombinant strains against different host bacteria

[0202]

[0203]

[0204] Example 7

[0205] Growth curve of Bacillus subtilis

[0206] 1. Strain resuscitation: Recombinant Bacillus subtilis rBS cotG-0859 The revival of wild-type Bacillus subtilis 168 was the same as that of the strain in Example 3(3).

[0207] 2. Sample preparation: Resuscitated recombinant Bacillus subtilis rBS was cultured in LB medium. cotG-0859 Dilute 100 times with wild-type Bacillus subtilis 168, add 100 μL to microplates, perform 5 replicates per group, and monitor for 24 h using an automated growth curve analyzer.

[0208] 3. Growth status of the strain: from Figure 6 It can be observed that the growth rate is relatively slow and metabolism is active in the first 2 hours. After 2 hours, the growth rate accelerates, and the bacteria grow at a stable geometric rate. A plateau is reached at 8 hours and maintained for 8 hours. After 18 hours, the bacterial count decreases somewhat, and then fluctuates. Overall, the expression of the prophage lyase Lys0859 does not affect the growth of Bacillus subtilis.

[0209] Example 8

[0210] Sporulation curve of Bacillus subtilis

[0211] 1. Resuscitation of the strain: Same as step 3 in Example 3.

[0212] 2. Preparation of DSM medium: Same as step 1 in implementation case 4.

[0213] 3. Determine the spore production curve: Resuscitate B. subtilis 168 and rBS... cotG-0859 The bacterial culture was inoculated into DSM medium at an inoculum of 1%, and cultured at 37°C with constant shaking at 180 rpm. Every 2 hours, the culture was taken out, diluted, and plated to calculate the total number of bacteria. The culture was then treated at 85°C for 5 minutes to kill the vegetative bacteria, and the culture was diluted and plated to calculate the number of spores formed.

[0214] 4. Sporulation curve results of Bacillus subtilis: From Figure 7 As shown in the total bacterial count graph (a), the total bacterial count continuously increases over time, continuing to increase even after 24 hours. (From...) Figure 7 As shown in Figure (b), spore formation begins at 10 hours and the total bacterial count continues to increase until 24 hours. Overall, wild-type Bacillus subtilis 168 and recombinant Bacillus subtilis rBS... cotG-0859 There was no significant difference in the growth and spore formation of the vegetative strains, indicating that the expression of the prophage lyase Lys0859 does not affect the growth of Bacillus subtilis.

[0215] Example 9

[0216] SDS-PAGE and Western blot analysis were used to examine the expression of fusion proteins in recombinant spores.

[0217] 1. Reagent Preparation: ① ST Buffer Preparation: 1% SDS, 50mM DTT. ② Destaining Solution Preparation: Measure 330mL methanol, 100mL glacial acetic acid, and 570mL deionized water to a final volume of 1L. ③ Electroporation Buffer Preparation: Weigh 3.027g Tris, 14.26g glycine, 200mL methanol, and dilute to a final volume of 1L with deionized water. ④ Loading Buffer: 60mL 1mol / L Tris-HCl (pH=6.8), 500mL 50% glycerol, 200mL 10% SDS, 50mL β-mercaptoethanol, 100mL 1% bromophenol blue, and 90mL deionized water. ⑤ Coomassie Brilliant Blue Staining Solution: 1g Coomassie Brilliant Blue G-250, 450mL methanol, 100mL glacial acetic acid, and dilute to a final volume of 1L with deionized water. ⑥PBST: Dissolve 8 g / L NaCl, 0.24 g / L KH2PO4, 0.2 g / L KCl, and 3.63 g / L Na2HPO4·12H2O in 800 mL of deionized water. Adjust the pH to 7.4 with HCl, add deionized water to a final volume of 1 L, and add 0.5 mL of Tween 20. ⑦Blocking buffer: Dissolve 1 g of 5% skim milk powder in 20 mL of PBST.

[0218] 2. Extraction of capsid protein: Take the purified spores from step 4 of Example 4, 5 mL of ST buffer and 1×10 8 CFU spores were mixed evenly and treated at 70℃ for 30 min, centrifuged at 12000 rpm for 10 min, and the supernatant was collected. After filtering the supernatant, the capsid protein was concentrated using an ultrafiltration tube.

[0219] 3. Gel preparation: Prepare the gels according to the instructions of the purchased polyacrylamide gel electrophoresis kit. The stacking gel and separating gel should both be 12%.

[0220] 4. Boiling: Add 5×SDS-PAGE Loading buffer to the capsid protein, vortex to mix, and boil in a metal bath at 95℃ for 10 min.

[0221] 5. Sample loading: Add the cooked sample sequentially to the sample loading wells of the swimming lane (two copies each), and allow the sample to migrate to the separating gel at 80V for 30 minutes. Then, apply 120V for 50 minutes and turn off the power.

[0222] 6. Staining: Gently immerse one portion of the colloid in Coomassie Brilliant Blue staining solution and remove it after staining for 1 hour.

[0223] 7. Decolorization: Gently immerse the colloid in the decolorization solution, changing the solution continuously, until protein bands appear on the colloid.

[0224] 8. Transfer: Peel off another portion of the colloid and transfer the capsid protein from the colloid to the PVDF membrane using an electrophoresis apparatus. After soaking the gel and PVDF membrane in electrophoresis buffer, place the PVDF membrane on top of the gel and roll it back and forth with a glass rod to remove air bubbles. Cover the outer layers with filter paper soaked in electrophoresis buffer, keeping them moist and free of air bubbles, and then place them in the electrophoresis apparatus. Turn on the power to start electrophoresis and transfer. After 1 hour, the transfer is complete, and the PVDF membrane is removed.

[0225] 9. Sealing: At room temperature, the PVDF membrane is transferred to the sealing solution and sealed for 2 hours.

[0226] 10. Incubation with primary antibody: Discard the blocking solution, wash once with TBST for 10 min, add 10 mL of diluted 0859 polyclonal antibody (1:5000), incubate at 37℃ for 2 h, wash three times with TBST for 5 min each time.

[0227] 11. Incubation with secondary antibody: Add 10 mL of diluted secondary antibody (1:5000) and incubate at room temperature for 1 h. Wash 3 times with TBST, 10 min each time.

[0228] 12. Color development: Follow the instructions for the DAB color development kit (Beyotime).

[0229] SDS-PAGE results showed that: lane 1 of recombinant Bacillus subtilis showed a specific band at 50 kDa; lane 2 of prokaryotic pCold-0859 protein showed a specific band at 28 kDa; lane 3 of wild-type recombinant Bacillus subtilis 168 did not show a specific band. Figure 8 .

[0230] Western blotting results showed that: lane 1 showed a specific band at 50 kDa for recombinant Bacillus subtilis; lane 2 showed a specific band at 50 kDa for prokaryotic expression of pCold-cotG-0859 protein; lane 3 showed a specific band at 28 kDa for prokaryotic expression of pCold-0859 protein; and lane 4 showed no specific band for wild-type Bacillus subtilis 168. Figure 9 .

[0231] The results above show that the target fusion protein was successfully expressed on the surface of the recombinant spores.

[0232] Example 11

[0233] Immunofluorescence analysis

[0234] 1. Reagent preparation: Antibody binding buffer: Add BSA and goat serum to PBS to a mass concentration of 5%.

[0235] 2. Add the purified spore suspension from step 4 of Example 8 to a 1.5 mL EP tube and centrifuge at 8000 rpm for 15 min at 4°C. Take 1 mL of antibody-binding buffer pre-cooled on ice to suspend the spore precipitate, block at 4°C for 2 h, centrifuge and discard the supernatant, add an appropriate amount of polyclonal antibody (Anti-0859 antibody) to dilute it to 1:400, and incubate on ice for 2 h.

[0236] 3. After incubation, wash the spores three times with PBS, centrifuge at 4500 mm for 10 min at 4°C, and carefully remove the supernatant.

[0237] 4. Add FITC-labeled goat anti-mouse IgG (secondary antibody) diluted appropriately with PBS to a dilution of 1:400, and incubate on ice for 2 hours.

[0238] 5. After incubation, wash the spores three times with PBS, centrifuge at 4500 rpm for 10 min at 4°C, and carefully remove the supernatant.

[0239] 6. After resuspending the spore precipitate in PBS, take 10 μL of the suspension and drop it onto a glass slide, cover it with a coverslip, and dry it in the dark. Then observe it immediately with a laser confocal microscope.

[0240] See results Figure 10 .Depend on Figure 10 It can be seen that, compared with the spores produced by the host bacteria, clear fluorescence can be observed on the surface of the recombinant spores, indicating that the surface of the recombinant spores expresses prophage lysin.

[0241] As can be seen from the results of the above embodiments, the present invention fuses the gene encoding the Bacillus anchoring protein and the gene encoding the prophage lysin Lys0859 to form a fusion protein, clones it into the pDG364 vector, and transforms the resulting pDG364-cotG-0859 into B. subitilis 168 competent cells to form recombinant Bacillus (see...). Figure 11 The recombinant Bacillus strain described herein successfully displays the prephage lyase Lys0859 gene on the spore surface during endophageal spore formation, along with the expression of anchoring proteins. The spores are resistant to high temperatures, strong acids and alkalis, and the gastrointestinal environment. Furthermore, it exhibits antibacterial effects against Gram-positive bacteria such as Streptococcus suis, Staphylococcus aureus, Listeria monocytogenes, and Clostridium perfringens, providing an alternative antibiotic for Streptococcus suis infections.

[0242] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a recombinant Bacillus displaying a prephage lysin of Streptococcus suis in the preparation of an antimicrobial agent or feed, wherein the antimicrobial agent is a Listeria monocytogenes inhibitor and / or a Clostridium perfringens inhibitor, and the recombinant Bacillus displays the prephage lysin Lys0859 on the surface of the Bacillus anchoring protein; The Bacillus anchoring protein is cotG protein; The amino acid sequence of the cotG protein is shown in SEQ ID NO:1; The amino acid sequence of the prephage lysin Lys0859 is shown in SEQ ID NO:2; The amino acid sequence of the fusion protein formed by the anchoring protein cotG and the prophage lysin Lys0859 is shown in SEQ ID NO:

3.

2. The application according to claim 1, characterized in that, The host strain of the recombinant Bacillus is the Bacillus subtilis 168 standard strain.

3. The application according to claim 1, characterized in that, The method for constructing the recombinant Bacillus includes the following steps: Prepare a fusion gene of Bacillus anchoring protein cotG and prophage lysin Lys0859; The fusion gene was cloned into a shuttle plasmid to obtain a recombinant plasmid; The recombinant plasmid was transformed into a Bacillus host bacterium to obtain recombinant Bacillus bacterium displaying a Streptococcus suis prophage lysin.

4. The application according to claim 3, characterized in that, The method for preparing the fusion gene of Bacillus anchoring protein and prophage lyase Lys0859 includes amplifying the coding gene of Bacillus anchoring protein cotG and the coding gene of prophage lyase Lys0859 respectively, and obtaining the fusion gene cotG-0859 by overlapping PCR amplification. Primers for amplifying the gene encoding the Bacillus anchoring protein cotG include a forward primer with a nucleotide sequence as shown in SEQ ID NO:4 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:5; Primers for amplifying the gene encoding the prephage lysin Lys0859 include a forward primer with a nucleotide sequence as shown in SEQ ID NO:6 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:

7. Primers used for overlap PCR amplification include a forward primer with a nucleotide sequence as shown in SEQ ID NO:4 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:

7.

5. The application according to claim 3 or 4, characterized in that, The fusion gene is integrated into the amylase gene in the host genome of Bacillus subtilis.

6. The use of recombinant spores produced by recombinant Bacillus in the application of claim 1 in the preparation of antibacterial agents or feed, wherein the antibacterial agent is a Listeria monocytogenes inhibitor and a Clostridium perfringens inhibitor.

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