Bacillus subtilis for recombinant expression of hermetia illucens HIAMP0438 gene and construction method thereof

Abstract

The invention discloses bacillus subtilis for recombinant expression of a hermetia illucens HIAMP0438 gene and a construction method of the bacillus subtilis, and belongs to the technical field of gene engineering. The hermetia illucens recombinant protein used in the invention is obtained by expression of bacillus subtilis WB800N transferred into a pHT43-HIAMP0438 high-efficiency expression vector, and the pHT43-HIAMP0438 high-efficiency expression vector is obtained by a homologous recombination technology, so that high-level expression of the HIAMP0438 gene is realized, the HIAMP0438 gene can be efficiently secreted out of cells, the preparation process of the recombinant protein is greatly simplified, and the production cost is reduced. According to the present invention, the recombinant protein can significantly inhibit the growth of Escherichia coli, Salmonella and Listeria monocytogenes, and the recombinant protein can be used as the animal health product, and can be used for preventing and treating pig and chicken intestinal diarrhea caused by Escherichia coli and Salmonella and food-borne digestive tract diseases caused by Listeria monocytogenes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a recombinant Bacillus subtilis expressing a gene of Hermetia illucens HIAMP0438 and a construction method thereof. BACKGROUND

[0002] Bacillus subtilis is a kind of commonly used probiotic bacteria for livestock and humans, and has been approved as a feed additive. Bacillus subtilis Compared with the E. coli expression system and the yeast expression system, the physiological characteristics, biochemical characteristics and genetic characteristics of Bacillus subtilis have been fully studied, which makes it a promising microbial cell factory developed by using emerging genetic manipulation tools. The WB800N strain is obtained by knocking out eight extracellular proteases (nprE, nprB, aprE, epr, mpr, bpr, vpr, and WprA) based on the BS168 engineering strain, so there is no need to worry about degradation problems, and the target protein can be directly secreted into the culture medium. Bacillus subtilis is a gram-positive bacterium, which has an inhibitory effect on other gram-positive bacteria in the environment, but does not have an inhibitory effect on gram-negative bacteria.

[0003] E. coli and Salmonella can cause diarrhea in the digestive tract of livestock, and the conventional Bacillus subtilis has weak inhibitory effect on E. coli and Salmonella, and it is difficult to treat diarrhea caused by gram-negative bacteria by killing E. coli and Salmonella.

[0004] Antibiotics can kill E. coli and Salmonella, but in the context of the ban on antibiotics in livestock breeding, the application of antibiotics is greatly limited, and it is urgent to develop natural biological technology products such as antibacterial peptides and traditional Chinese medicines to kill E. coli and Salmonella as an alternative to antibiotics.

[0005] Antibacterial peptides refer to a kind of basic polypeptide substances with antibacterial activity induced in insects, with a molecular weight of about 2000-7000 Da, composed of 20-60 amino acid residues. Most of these active polypeptides have strong alkalinity, thermal stability and broad-spectrum antibacterial properties.

[0006] Hermetia illucens is a globally distributed resource insect that can effectively convert organic waste such as kitchen waste and livestock manure into biomass and compost, which can be used as animal feed and biofertilizer. Hermetia illucens contains more than 50 antibacterial peptide genes, and the antibacterial peptides produced in the body have high antibacterial capacity.

[0007] HIAMP0438 belongs to a class of arginine-rich non-glycosylated cationic peptides expressed by Hermetia illucens, which was first induced from the hemolymph of Hermetia illucens larvae after immunization and purified by solid phase extraction and reverse phase chromatography, and the molecular weight thereof is 21.71 kDa. It is not clear whether HIAMP0438 has the function of inhibiting gram-negative bacteria.

[0008] Although the Hermetia illucens antibacterial peptide has good inhibitory effect on gram-negative bacteria such as Escherichia coli, it is difficult to breed a large number of Hermetia illucens as insects, and how to obtain a large amount of Hermetia illucens antibacterial peptide is an important biotechnological problem.

[0009] Bacillus subtilis has no significant codon bias and is one of the excellent choices for expressing foreign proteins. Efficient expression of Hermetia illucens antibacterial peptide gene in Bacillus subtilis is a potential important way to obtain Hermetia illucens antibacterial peptide protein for treating digestive tract diseases caused by gram-negative bacteria such as Escherichia coli and foodborne diseases caused by Listeria monocytogenes. The conventional technology for expressing heterologous proteins using Bacillus subtilis as the starting strain is to use a free plasmid to carry foreign genes for heterologous protein expression in bacteria. At present, there is no related report on obtaining a large amount of Hermetia illucens antibacterial peptide by using this technology. SUMMARY

[0010] The purpose of the present application is to provide a recombinant Bacillus subtilis expressing Hermetia illucens HIAMP0438 gene and a construction method thereof to solve the problems existing in the prior art. The present application uses a Bacillus subtilis high expression vector, the target gene is connected by homologous recombination technology, and IPTG is used for induction expression, so as to realize the efficient and stable expression of HIAMP0438 gene in Bacillus subtilis, and determine that the recombinant strain has the new function of inhibiting Escherichia coli and Salmonella. The recombinant strain can be used as a feed additive for diarrhea diseases of livestock and poultry caused by Escherichia coli, Salmonella and the like.

[0011] To achieve the above purpose, the present application provides the following solutions: One of the technical solutions of the present application is a recombinant expression vector of Hermetia illucens antibacterial peptide HIAMP0438 gene, which is constructed with pHT43 as the backbone and contains the nucleotide sequence shown in SEQ ID NO. 4.

[0012] The second technical solution of the present application is a recombinant Bacillus subtilis of Hermetia illucens antibacterial peptide HIAMP0438 gene, which uses Bacillus subtilis WB800N as the starting strain and contains the recombinant expression vector.

[0013] The third technical solution of the present application is the application of the recombinant expression vector or the recombinant Bacillus subtilis in preparing Hermetia illucens antibacterial peptide HIAMP0438.

[0014] The fourth technical solution of the present invention is a method for preparing the black soldier fly antimicrobial peptide HIAMP0438, which utilizes the recombinant Bacillus subtilis to produce the black soldier fly antimicrobial peptide HIAMP0438 under the induction of isopropyl-β-D-thiogalactoside.

[0015] The fifth technical solution of the present invention is the application of the recombinant Bacillus subtilis in the preparation of drugs for preventing and treating diseases caused by Gram-negative bacteria.

[0016] The sixth technical solution of the present invention is a drug for preventing and treating diseases caused by Gram-negative bacteria, comprising the recombinant Bacillus subtilis.

[0017] Based on the above technical solution, the present invention has the following technical effects: This invention achieves the secretory expression of the antimicrobial peptide HIAMP0438. The expression product is directly secreted extracellularly without further purification, and the recombinant strain has been confirmed to possess a novel function of inhibiting Escherichia coli and Salmonella. Furthermore, the expression host selected in this invention is a recognized probiotic that is non-pathogenic to animals, thus realizing the combined application of probiotics and the antimicrobial peptide. Attached Figure Description

[0018] Figure 1 This is a PCR amplification of the signal peptide-black soldier fly antimicrobial peptide HIAMP0438 ligation fragment. M: TakaraDL5000 DNA Marker; A: negative control, plasmid pHT43; B: blank control, H2O; 1–5: target fragment DNA amplification product (630 bp).

[0019] Figure 2 The results show the enzyme digestion of the pHT43 plasmid. M: TakaraDL15000 DNA Marker; 1: pHT43 plasmid; 2-5: pHT43 plasmid BamHI digestion products.

[0020] Figure 3 PCR validation of the recombinant vector pHT43-HIAMP0438. M: TakaraDL5000 DNA Marker; A: Positive control, pUC-HIAMP0438 plasmid; B: Negative control, E. coli culture; C: Blank control, H2O; 1-4: Positive recombinants transformed from TOP10 competent cells by the recombinant plasmid.

[0021] Figure 4The results show the double digestion of the recombinant vector pHT43-HIAMP0438 with BamHI and Xbal. M: TakaraDL10000 DNA Marker; 1: pHT43-HIAMP0438 recombinant vector; 2: pHT43-HIAMP0438 recombinant vector double digestion product.

[0022] Figure 5 Sequencing alignment of the recombinant vector pHT43-HIAMP0438.

[0023] Figure 6 The results of electroporation of Bacillus subtilis competent cells on plate culture.

[0024] Figure 7 The results are PCR identification results of Bacillus subtilis transformed with recombinant plasmids. M: TakaraDL2000 DNA Marker; A: Positive control, pHT43-HIAMP0438 recombinant vector; B: Negative control, Bacillus subtilis WB800N; C: Blank control, H2O; 1-4: Genomic DNA amplification products (630 bp).

[0025] Figure 8 For the determination of the growth curve of recombinant bacteria.

[0026] Figure 9 The results show the stability of the HIAMP0438 gene after passage of the recombinant bacteria. M: TakaraDL2000 DNA Marker; A: Positive control, pHT43-HIAMP0438: recombinant vector; B: Negative control, Bacillus subtilis WB800N; C: Blank control, H2O; 1-9: bacterial suspensions of the recombinant bacteria at generations 1, 5, 10, 15, 20, 25, 30, 35, and 40.

[0027] Figure 10 This study analyzed the concentration and timing of HIAMP0438 mRNA expression in recombinant bacteria induced by IPTG. Note: Different letters indicate significant differences between groups (P < 0.05, n = 3).

[0028] Figure 11 SDS-PAGE identification results of recombinant proteins expressed by recombinant Bacillus subtilis. In the table, M: 2-40 Marker Protein marker; 1: Fermentation broth for induced expression; 2: Uninduced fermentation broth; 3: Fermentation broth with pHT43 empty vector; 4: Ultrasonic precipitation for induced expression; 5: Ultrasonic precipitation for uninduced expression; 6: Ultrasonic precipitation with pHT43 empty vector; 7: Concentrated supernatant for induced expression; 8: Concentrated supernatant for uninduced expression; 9: Concentrated supernatant for pHT43 empty vector.

[0029] Figure 12 Analysis of the relative expression level of HIAMP0438 protein.

[0030] Figure 13 The inhibitory effect of recombinant bacteria on Escherichia coli was studied. The components included: 1: ampicillin sodium indicator; 2: 2×YT liquid culture medium; 3: concentrated supernatant of Bacillus subtilis WB800N bacterial culture; 4: sonication of Bacillus subtilis WB800N bacterial cell precipitation; 5: concentration of supernatant from recombinant bacterial induction culture; and 6: sonication of recombinant bacterial induction bacterial cell precipitation.

[0031] Figure 14 The inhibitory effect of recombinant bacteria on Salmonella was demonstrated. The components included: 1: ampicillin sodium indicator; 2: 2×YT liquid culture medium; 3: concentrated supernatant of Bacillus subtilis WB800N bacterial culture; 4: sonication of Bacillus subtilis WB800N bacterial cell precipitation; 5: concentration of supernatant from recombinant bacterial induction culture; and 6: sonication of recombinant bacterial induction bacterial cell precipitation.

[0032] Figure 15 The inhibitory effect of recombinant bacteria on Listeria monocytogenes was investigated. The components included: 1: ampicillin sodium indicator; 2: 2×YT liquid culture medium; 3: concentrated supernatant of Bacillus subtilis WB800N bacterial culture; 4: sonication of Bacillus subtilis WB800N bacterial cell precipitation; 5: concentration of supernatant from recombinant bacterial induction culture; and 6: sonication of recombinant bacterial induction bacterial cell precipitation.

[0033] Figure 16 The effect of recombinant bacteria on the growth curve of Escherichia coli.

[0034] Figure 17 The effect of recombinant bacteria on the growth curve of Salmonella.

[0035] Figure 18 The effect of recombinant bacteria on the growth curve of Listeria. Detailed Implementation

[0036] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0037] This invention provides a recombinant expression vector for the black soldier fly antimicrobial peptide HIAMP0438 gene, constructed with pHT43 as the backbone, and containing the nucleotide sequence shown in SEQ ID NO.4.

[0038] This invention also provides a recombinant Bacillus subtilis strain containing the black soldier fly antimicrobial peptide HIAMP0438 gene, with Bacillus subtilis WB800N as the starting strain, and containing the recombinant expression vector described above.

[0039] This invention also provides the application of the recombinant expression vector or the recombinant Bacillus subtilis in the preparation of the black soldier fly antimicrobial peptide HIAMP0438.

[0040] In some specific embodiments, the amino acid sequence of the black soldier fly antimicrobial peptide HIAMP0438 is shown in SEQ ID NO.1.

[0041] This invention also provides a method for preparing the black soldier fly antimicrobial peptide HIAMP0438, which utilizes the recombinant Bacillus subtilis to produce the black soldier fly antimicrobial peptide HIAMP0438 under the induction of isopropyl-β-D-thiogalactoside.

[0042] In some specific embodiments, the concentration of the isopropyl-β-D-thiogalactoside in the culture medium is 1 mmol / L.

[0043] This invention also provides the application of the recombinant Bacillus subtilis in the preparation of drugs for preventing and treating diseases caused by Gram-negative bacteria.

[0044] In some specific implementations, the Gram-negative bacteria include Escherichia coli, Salmonella, and Listeria monocytogenes.

[0045] This invention also provides a drug for preventing and treating diseases caused by Gram-negative bacteria, including the recombinant Bacillus subtilis.

[0046] The Bacillus subtilis expression system constructed in this invention is safe and non-toxic, and can efficiently secrete recombinant black soldier fly antimicrobial peptide proteins. The recombinant black soldier fly protein used in this invention is obtained by expressing Bacillus subtilis WB800N cells transformed with the pHT43-HIAMP0438 high-efficiency expression vector. This pHT43-HIAMP0438 high-efficiency expression vector is obtained through homologous recombination technology, achieving not only high-level expression of the HIAMP0438 gene but also efficient extracellular secretion, greatly simplifying the recombinant protein preparation process and facilitating large-scale production. This recombinant protein significantly inhibits the growth of Escherichia coli, Salmonella, and Listeria monocytogenes. This recombinant bacteria can be used as an animal health product for the prevention and treatment of intestinal diarrhea in pigs and chickens caused by Escherichia coli and Salmonella, and foodborne digestive tract diseases caused by Listeria monocytogenes.

[0047] The HIAMP0438 gene, derived from the black soldier fly, encodes a recombinant protein with antibacterial activity. The HIAMP0438 protein significantly inhibits the growth of *Escherichia coli*, *Salmonella*, and *Listeria monocytogenes*. HIAMP0438 protein can be used as an animal health supplement for the prevention and treatment of intestinal diarrhea in pigs and chickens caused by *E. coli* and *Salmonella*, and foodborne digestive tract diseases caused by *Listeria monocytogenes*.

[0048] The HIAMP0438 protein has the amino acid sequence SEQ ID NO:1 (NCBI Genebank Accession: XP_037916366.1, 209aa). The HIAMP0438 protein is a mature peptide of the antimicrobial peptide (NCBI Genebank Accession: XM_038060438.1, 630bp) with codon-optimized sequence containing a transcription terminator sequence, as shown in SEQ ID NO.4.

[0049] Electroporation using the recombinant expression vector pHT43-HIAMP0438 Bacillus subtilis WB800N was further cultured in chloramphenicol-containing 2×YT liquid medium and agar medium, and the pHT43-HIAMP0438 gene sequence was amplified by PCR to screen for positive transformed clones.

[0050] This invention establishes the optimal concentration and induction time for IPTG substrate to induce expression of the HIAMP0438 gene: 1 mmol / L IPTG, induced at 37°C for 12 h.

[0051] This invention achieves the secretory expression of the antimicrobial peptide HIAMP0438, with the amino acid sequence SEQ ID NO.1. After protein expression, it is distributed extracellularly, achieving high-level secretory expression.

[0052] In addition, previous studies have shown that target proteins with large tags do not have antibacterial activity and need to be removed, which reduces protein yield. This invention can concentrate recombinant proteins without using purification tags, which facilitates subsequent purification and does not require tag removal while still maintaining antibacterial activity.

[0053] This invention provides an engineered Bacillus subtilis strain expressing HIAMP0438. The engineered strain is obtained by cloning the gene sequence encoding the HIAMP0438 fusion protein into an expression vector after codon optimization, thus obtaining a recombinant expression vector. The recombinant expression vector is then transformed into Bacillus subtilis, and positive transformants are obtained by screening, which are the engineered Bacillus subtilis strains.

[0054] The expression vector can be pHT43, etc.

[0055] This invention provides a method for constructing an engineered Bacillus subtilis strain expressing the black soldier fly HIAMP0438 gene, comprising the following steps: (1) The target gene sequence shown in SEQ ID NO:4 was ligated into the linearized expression vector pHT43 by homologous recombination technology to obtain the recombinant expression vector; (2) The recombinant expression vector was transformed into Bacillus subtilis, and the positive transformants obtained by screening were the Bacillus subtilis engineered bacteria.

[0056] The Bacillus subtilis can be WB800N, etc.

[0057] This invention relates to a recombinant Bacillus subtilis strain expressing the black soldier fly HIAMP0438 gene and its construction method: using homologous recombination, the coding region of the black soldier fly HIAMP0438 gene is cloned into the Bacillus subtilis expression vector pHT43, and recombinant Bacillus subtilis strain expressing HIAMP0438 protein and significantly inhibiting the growth of Escherichia coli, Salmonella and Listeria monocytogenes is obtained by IPTG induction.

[0058] The vector pHT43 and Bacillus subtilis WB800N used in the following examples were purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0059] Example 1 Method for constructing Bacillus subtilis engineered strain expressing the black soldier fly HIAMP0438 gene 1. Construction of recombinant expression vectors (1) Synthesis of the black soldier fly HIAMP0438 gene and construction of the cloning vector In this embodiment, the black soldier fly HIAMP0438 gene (NCBI Genebank Accession: XM_038060438) was amplified by PCR to determine the nucleotide sequence. The nucleotide sequence has a Bamh I restriction site at the 5' end and an Xbal restriction site at the 3' end. It was then ligated into the cloning vector pUC57 to form the recombinant vector pUC57-HIAMP0438. The sequence was chemically synthesized by Wuhan Qingke and has a total length of 772 bp.

[0060] The HIAMP0438 gene sequence was amplified in the PCR reaction system shown in Table 1 and then recovered by gel extraction.

[0061] Table 1. PCR reaction system for amplifying the HIAMP0438 gene sequence.

[0062] The primer sequences are as follows: HIAMP0438-F:5'-aaaacatcagccgtaGGATCCATGGCGCTGACCCGAACTT-3' (underlined area is BamHI recognition site); HIAMP0438-R:5'-gacgtcgactctaga GGATCC CTAAAAACGATGAGAAAACCCCA-3' (The underlined part is the BamHI recognition site).

[0063] The reaction conditions were: 94℃ pre-denaturation for 5 min, 32 cycles (94℃ denaturation for 30 s, 53℃ annealing for 30 s, 72℃ extension for 37 s), and a final extension at 72℃ for 10 min. The obtained PCR products were detected by gel electrophoresis using 1.5% agarose gel. The results are as follows: Figure 1 As shown, PCR amplification products with the same size as the target sequence (772bp) were obtained.

[0064] The HIAMP0438 gene sequence (containing the HIAMP0438 structural gene sequence and a partial pHT43 vector backbone sequence for seamless cloning to construct a recombinant vector) is shown as SEQ ID NO.2: aatttattcacttattagtattaacctaacgaggaaagtattttgtattaaaagacgcaatggcattgacaaggacatcgatgattattgcaattgttgcaatctgcggtacttcaacattgggccaactatcaggatcgattacacccgatatggcaggaggcaataatgttaacatcatggcatcgaaattcttgggaaaccccaatcacaacattggtggaggagttttcgccgcaggcaacacacgatccaataccccatccttgggagcttttggaaccttgaacctaaaagaccatagtctgggggtgtcgaaaaccatcactccaggggtaagtgatacattcagtcaaaatgcacgactaaatatcctgaagactcctgatcatcgagtggacgcgaacgttttcaatagtcatacaaggctgaataatggattcgcattcgataaacgaggcggtagtttggattatacccacagggcaggacatggtctttctttgggagccagtcatatccctaaatttggaacgactgctgaattaacgggcaaagctaacctctggagatcacccagtggtctatcaacttttgatttaactggaagtgcatcgagaacgtttggtggaccaatggctggtagaaataattttggtgcaggtttaggatttagccataggttttagtgaaaatctatagaataattcgttattgttaaattatttttaatacaacaaaagtctggagaatatattgcataaaagatcaa。

[0065] The gene sequence of Hermetia illucens HIAMP0438 (NCBI Genebank Accession: XM_038060438, 630bp) is shown in SEQ ID NO.3: ATGGCATTGACAAGGACATCGATGATTATTGCAATTGTTGCAATCTGCGGTACTTCAACATTGGGCCAACTATCAGGATCGATTACACCCGATATGGCAGGAGGCAATAATGTTAACATCATGGCATCGAAATTCTTGGGAAACCCCAATCACAACATTGGTGGAGGAGTTTTCGCCGCAGGCAACACACGATCCAATACCCCATCCTTGGGAGCTTTTGGAACCTTGAACCTAAAAGACCATAGTCTGGGGGTGTCGAAAACCATCACTCCAGGGGTAAGTGATACATTCAGTCAAAATGCACGACTAAATATCCTGAAGACTCCTGATCATCGAGTGGACGCGAACGTTTTCAATAGTCATACAAGGCTGAATAATGGATTCGCATTCGATAAACGAGGCGGTAGTTTGGATTATACCCACAGGGCAGGACATGGTCTTTCTTTGGGAGCCAGTCATATCCCTAAATTTGGAACGACTGCTGAATTAACGGGCAAAGCTAACCTCTGGAGATCACCCAGTGGTCTATCAACTTTTGATTTAACTGGAAGTGCATCGAGAACGTTTGGTGGACCAATGGCTGGTAGAAATAATTTTGGTGCAGGTTTAGGATTTAGCCATAGGTTTTAG。

[0066] The HIAMP0438 gene sequence was codon optimized, as shown in SEQ ID NO.4.

[0067] (2) Expression vector extraction and enzyme digestion: E. coli containing the expression vector pHT43 were streaked onto 2×YT agar plates for activation. The next day, single colonies were inoculated into 20 mL of 2×YT liquid medium containing 100 μg / mL ampicillin and cultured overnight at 37°C and 200 rpm. Plasmids were then extracted. The extracted plasmids were digested with single enzymes for subsequent homologous recombination. The enzyme digestion system is shown in Table 2.

[0068] Table 2 Single enzyme digestion reaction system of recombinant plasmid

[0069] The enzyme digestion conditions were: 37℃ in a metal bath for 45 min, with plasmid DNA at pH T43. The above reagents were added to the PCR reaction tube, mixed well, and briefly centrifuged. After incubation at 37℃ for 45 min, the enzyme digestion was complete. All reaction solutions were added to 10 μL of 6× loading buffer and mixed thoroughly. Electrophoresis was performed using 1% agarose gel. After successful electrophoresis, the target band (8057 bp) was excised from the gel and recovered for subsequent recombination reactions. The detection results are as follows: Figure 2 As shown. Figure 2 In the diagram, 1 represents the plasmid before enzyme digestion (with different conformations and 3 bands), and 2-5 represent the linearized plasmids after enzyme digestion, indicating that the vector digestion was successful and it can be used to construct recombinant vectors.

[0070] (3) The HIAMP0438 gene was ligated into the expression vector: HIAMP0438 was ligated to the linearized expression vector pHT43 using homologous recombination technology. The ligation reaction system is shown in Table 3.

[0071] Table 3 Connection Reaction System

[0072] The homologous recombination reaction was carried out at 50°C for 5 min. The ligation product was then transformed into the TOP10 competent cloning vector and plated on 2×YT agar plates containing 100 μg / mL ampicillin resistance. The cells were cultured overnight, and single colonies were picked for PCR, enzyme digestion, and sequencing verification. The PCR reaction system is shown in Table 1; the enzyme digestion system is shown in Table 4.

[0073] Table 4 Double enzyme digestion system

[0074] The results of enzyme digestion and sequencing of recombinant plasmid pHT43-HIAMP0438 are as follows: Figure 4 , Figure 5 As shown in the figure. Enzyme digestion results indicate that the recombinant plasmid contains the HIAMP0438 gene sequence, and sequencing alignment shows that the sequenced genome and the HIAMP0438 sequence have 100% homology.

[0075] Example 2 Electroporation of Bacillus subtilis using recombinant expression vector pHT43-HIAMP0438 and screening of positive electroporation clones Preparation of competent cells of Bacillus subtilis: Bacillus subtilis WB800N was streaked onto 2×YT solid medium plates in a clean bench and incubated upside down in a 37°C water-jacketed incubator for 12 hours. A single colony of Bacillus subtilis from the plate was inoculated into an Erlenmeyer flask containing 5 mL of 2×YT liquid medium and incubated overnight at 37°C with a shaker at 200 rpm. 2.5 mL of the cultured bacterial solution was inoculated into an Erlenmeyer flask containing 40 mL of growth medium and incubated at 37°C with a shaker at 200 rpm for 4–5 hours. The cultured bacterial solution was transferred to two sterile 50 mL centrifuge tubes, incubated on ice for 10 min, and then centrifuged at 6000 g at 4°C for 10 min in a centrifuge pre-chilled at 4°C. The supernatant in the 50 mL centrifuge tubes was carefully discarded, and the remaining solution was gently resuspended in 25 mL of pre-chilled electroporation wash buffer and centrifuged at 6000 g at 4°C for 10 min in a centrifuge pre-chilled at 4°C. This process was repeated four times. Carefully discard the supernatant in the 50 mL centrifuge tube, gently resuspend the precipitate with 1 mL of electroporation wash buffer, and aliquot 60 μL into a 1.5 mL EP tube and store at -80°C.

[0076] The recombinant expression vector pHT43-HIAMP0438 was electrotransformed into competent Bacillus subtilis cells: Remove Bacillus subtilis competent cells from the -80℃ freezer and thaw on ice. Add 3-5 μL of the recombinant expression vector pHT43-HIAMP0438 to the dispensed competent cells, gently mix with a pipette tip, and incubate on ice for 10 min. Transfer the liquid from the tube to a clean, UV-sterilized electroporation cuvette. Turn on the electroporator, wipe the cuvette clean, and place it in the electroporator. Set the voltage to 2600 V, resistance to 200 Ω, capacitance to 25 μF, and time to 4 ms, and begin electroporation. After electroporation, quickly add 1 mL of pre-chilled recovery solution to the cuvette, mix with a pipette tip, transfer to a clean 1.5 mL EP tube, and incubate at 37℃ on a shaker at 200 rpm for 4 h. Centrifuge the culture after 4 h at 4000 rpm for 5 min, and discard 900 μL of supernatant. Resuspend the precipitate in the remaining supernatant and spread it evenly on 2×YT agar plates with a final chloramphenicol concentration of 15 μg / mL. Incubate the plates upright at 37°C for 30 min until the bacterial culture is completely absorbed. Then invert the plates and incubate overnight. The transformation results are as follows. Figure 6 As shown in the figure, the results indicate that positive transformed colonies were successfully obtained.

[0077] Identification of electroporated Bacillus subtilis: Positive strains from electroporation plates were transferred to LB liquid medium with a final chloramphenicol concentration of 15 μg / mL and cultured at 37°C with a shaker at 180 r / min for 12 h. Using the bacterial culture as a template, primers were designed to amplify the HIAMP0438 gene sequence via PCR. The PCR-positive bacterial cultures were retained.

[0078] Table 5. PCR system for electroporation identification of Bacillus subtilis

[0079] The results are as follows Figure 7 As shown in Table 5, the PCR reaction system contains the target fragment (630bp) in the obtained single colony, proving that the recombinant expression vector pHT43-HIAMP0438 has been successfully transformed into Bacillus subtilis.

[0080] Example 3 Growth curves and exogenous gene stability analysis of recombinant Bacillus subtilis Growth curve determination of recombinant bacteria: After inoculating Bacillus subtilis WB800N and recombinant bacterial cultures respectively, the cultures were shaken and cultured. OD was measured every 2 h. 600 Absorbance values ​​were used to analyze the growth curves of the two bacteria over 24 hours. Figure 8 The results showed that there was no significant difference in growth performance between the recombinant bacteria and the WB800N strain.

[0081] Results of HIAMP0438 gene stability assay after recombinant bacterial passage: The recombinant bacteria were still able to amplify the HIAMP0438 gene fragment after 40 passages. Figure 9 Furthermore, the sequencing results of the HIAMP0438 fragment amplified in the 40th generation were consistent with the target sequence, indicating that the recombinant bacteria have gene stability and recombination stability.

[0082] Example 4 Establishment of conditions for inducing recombinant Bacillus subtilis HIAMP0438 gene expression Concentration and timing analysis of HIAMP0438 gene mRNA expression in recombinant bacteria induced by isopropyl-β-D-thiogalactopyranoside (IPTG): Culture recombinant Bacillus subtilis to its OD 600 When the value was in the range of 0.6~0.8, 1 mmol / L IPTG was added to induce expression. RNA was extracted from 1 mL of bacterial culture at 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h and reverse transcribed into cDNA. After dilution 10-fold, qPCR was performed using 16S rRNA of Bacillus subtilis WB800N as an internal reference to obtain the ΔCT value of the HIAMP0438 gene. The experimental groups were those with added inducer, and the control group was the same as the experimental groups but without added inducer at the same time. ΔCT = target gene CT value - mean CT value of internal reference gene; ΔΔCT = experimental group ΔCT - control group ΔCT. The 2-1 values ​​were calculated. -ΔΔCT For the expression quantity.

[0083] Depend on Figure 10As shown, the expression levels of the HIAMP0438 gene in recombinant Bacillus subtilis induced by IPTG at the same concentration but different induction times differed significantly from those in uninduced Bacillus subtilis. At 12 h of induction, the HIAMP0438 gene mRNA expression level induced by 1 mmol / L IPTG was approximately 13-fold higher than that in uninduced HIAMP0438 gene mRNA. This concentration and induction time were selected as the optimal IPTG induction conditions for subsequent protein expression analysis and antibacterial experiments.

[0084] Example 5 Analysis of the induced expression of protein from recombinant Bacillus subtilis HIAMP0438 Analysis of protein expression levels encoded by the HIAMP0438 gene in IPTG-induced recombinant bacteria: (1) Recombinant Bacillus subtilis and Bacillus subtilis transfected with the pHT43 empty vector plasmid were streaked onto 2×YT agar plates containing chloramphenicol at a final concentration of 15 μg / mL, and cultured at 37℃ for 12 h in a water-insulated incubator. (2) Single colonies of activated recombinant Bacillus subtilis and Bacillus subtilis transfected with the pHT43 empty vector plasmid were picked from the plates and transferred to 5 mL of sterile 2×YT liquid medium (chloramphenicol concentration 15 μg / mL), and cultured at 37℃ and 180 r / min for 12 h in a shaker.

[0085] The recombinant bacterial culture from (2) was inoculated at 1% in 30 mL of sterile 2×YT liquid medium (chloramphenicol concentration 15 μg / mL), and cultured at 37℃ on a shaker at 180 r / min. When the OD value of the bacterial culture reached 0.6~0.8, 1 mmol / L of IPTG was added, and the culture was cultured at 37℃ on a shaker at 180 r / min for 12 h. The Bacillus subtilis culture from (2) with pHT43 empty vector plasmid was inoculated at 1% in 30 mL of sterile 2×YT liquid medium (chloramphenicol concentration 15 μg / mL), and cultured at 37℃ on a shaker at 180 r / min for 12 h.

[0086] Ultrasonic disruption: (1) The induced and uninduced bacterial cultures of recombinant Bacillus subtilis and the bacterial culture of Bacillus subtilis transfected with the pHT43 empty vector plasmid were transferred to 50 mL centrifuge tubes, respectively. The tubes were centrifuged at 4200 rpm for 30 min in a pre-cooled 4℃ centrifuge and the supernatant was collected. (2) The bacterial supernatant was transferred to a clean 15 mL ultrafiltration tube and centrifuged at 4200 rpm for 10 min in a 4℃ centrifuge to concentrate the supernatant. The concentrated supernatant was placed in a 10 mL EP tube and stored in a -4℃ refrigerator. The precipitate in each tube was resuspended in 4 mL PBS to OD. 600 The value was 2.0, and the sample was placed on ice. Ultrasonic disruption was performed using an ultrasonic disruptor.

[0087] Protein sample preparation: Take 80 µL each of the supernatant concentrate from the uninduced and induced recombinant bacteria, as well as the supernatant from Bacillus subtilis transformed with the pHT43 empty vector plasmid, the sonicated precipitate, and the fermentation broth, and add 20 µL of 5× loading buffer. Vortex to mix. Incubate at 100 °C in a metal bath for 10 min, and then store at -20 °C.

[0088] SDS-PAGE protein gel preparation and electrophoresis: Preparation and electrophoresis of small molecule protein gels: After washing and drying the glass plates for gel preparation, fix them in place and check that the bottom is sealed. Use the Sangon Tricine-SDS-PAGE small molecular weight protein gel preparation kit to prepare a 16.5% separating gel and a 4% stacking gel. Fix the prepared gels in the electrophoresis tank, remove the comb, add 1× diluted cathodic electrophoresis buffer between the two gels, and add 1× diluted anodic electrophoresis buffer next to the two gels. Place the electrophoresis tank in a 4°C refrigerator, pre-run at 30 V for 10 min, and then load the samples. Load 5 µL of Seville small molecule protein marker, and load 20 µL of each sample. Adjust the voltage to 150 V and electrophores until the bromophenol blue indicator just runs out of the gel edge.

[0089] Coomassie Brilliant Blue staining: Cut the gel, add water and heat in a microwave oven on medium heat for 3 minutes. Then transfer the gel to a petri dish, add 50 mL of Coomassie Brilliant Blue rapid staining solution and stain for 15 minutes. Add water to destain and take pictures for observation.

[0090] Results analysis: SDS-PAGE was used to detect the expression of HIAMP0438 protein (21.7 kDa) in recombinant Bacillus subtilis under optimal IPTG induction conditions (see [link to results]). Figure 11 and 12 The supernatant and concentrate of recombinant Bacillus subtilis culture induced by IPTG showed a newly added high-expression protein band at 21.7 kDa compared to uninduced recombinant Bacillus subtilis and Bacillus subtilis with empty vector, demonstrating that IPTG-induced recombinant Bacillus subtilis expressed the HIAMP0438 target protein and that the protein was secreted extracellularly. The cell lysate of recombinant Bacillus subtilis induced by IPTG did not show a newly added high-expression protein band at 21.7 kDa compared to uninduced recombinant Bacillus subtilis and Bacillus subtilis with empty vector, demonstrating that the HIAMP0438 target protein was secreted extracellularly and not present intracellularly after induction.

[0091] The sequence of HIAMP0438 protein is as SEQ ID Shown in NO.1: MALTRTSMIIAIVAICGTSTLGQLSGSITPDMAGGNNVNIMASKFLGNPNHNIGGGVFAAGNTRSNTPSLGAFGTLNLKDHSLGVSKTITPGVSDTFSQNARLNILKTPDHRVDANVFNSHTRLNNGFAFDKRGGSLDYTHRAGHGLSLGASHIPKFGTTAELTGKANLWRSPSGLSTFDLTGSASRTFGGPMAGRNNFGAGLGFSHRF.

[0092] Example 6 Analysis of the inhibitory effects of recombinant Bacillus subtilis on three enteropathogenic bacteria Inhibitory effect of recombinant bacteria on Escherichia coli (O157:H7, ATCC35150): The Oxford cup method (6 mm inner diameter, 8 mm outer diameter) was used, with *E. coli* as the indicator bacterium. After 24 h of incubation, the results showed that ampicillin indicator had an inhibition zone with a radius of approximately 15 mm. The supernatant of *Bacillus subtilis* WB800N and the precipitate of *Bacillus subtilis* WB800N were sonicated, and the supernatant of the recombinant bacteria was induced by 1 mmol / L IPTG. The supernatant of the recombinant bacteria precipitated and was sonicated again. The supernatant of the recombinant bacteria induced by 1 mmol / L IPTG showed an inhibition zone. Figure 13 The radius of the inhibition zone is approximately 12mm.

[0093] The sonicated growth curves of the recombinant bacteria supernatant and precipitate induced by the addition of 1 mmol / L IPTG were significantly slower than those induced by 2×YT culture. Figure 16 The results showed that the recombinant bacteria had an inhibitory effect on the growth of Escherichia coli.

[0094] Inhibitory effect of recombinant bacteria on Salmonella (CmCC(B)50335): The Oxford cup method (6 mm inner diameter, 8 mm outer diameter) was used, with Salmonella as the indicator bacterium. After 24 h of incubation, the results showed that ampicillin indicator had an inhibition zone with a radius of approximately 12 mm. The supernatant of Bacillus subtilis WB800N and the precipitate of Bacillus subtilis WB800N were sonicated, and the supernatant of recombinant bacteria was induced by 1 mmol / L IPTG. The supernatant of recombinant bacteria precipitated and was sonicated again. The supernatant of recombinant bacteria induced by 1 mmol / L IPTG showed an inhibition zone. Figure 14 The inhibition zone radius was approximately 9 mm. The results indicate that the recombinant bacteria inhibited the growth of Salmonella.

[0095] The sonicated growth curves of the recombinant bacteria supernatant and precipitate induced by the addition of 1 mmol / L IPTG were significantly slower than those induced by 2×YT culture. Figure 17 The results showed that the recombinant bacteria had an inhibitory effect on the growth of Salmonella.

[0096] Inhibitory effect of recombinant bacteria on Listeria monocytogenes (10403s, serotype 1 / 2a, ATCC 19111): The Oxford cup method (6 mm inner diameter, 8 mm outer diameter) was used, with *Listeria monocytogenes* as the indicator bacterium. After 24 hours of incubation, the results showed that ampicillin indicator exhibited an inhibition zone with a radius of approximately 22 mm. The supernatant of *Bacillus subtilis* WB800N and the precipitate of *Bacillus subtilis* WB800N were sonicated, and the supernatant of recombinant bacteria was induced with 1 mmol / L IPTG. The supernatant of the recombinant bacteria also exhibited an inhibition zone after sonication. Figure 15 The inhibition zone radius was approximately 20 mm. The results indicate that the recombinant bacteria inhibited the growth of Listeria monocytogenes.

[0097] The sonicated growth curves of the recombinant bacteria supernatant and precipitate induced by the addition of 1 mmol / L IPTG were significantly slower than those induced by 2×YT culture. Figure 18 The results showed that the recombinant bacteria had an inhibitory effect on the growth of Listeria monocytogenes.

[0098] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A recombinant expression vector of the black soldier fly antimicrobial peptide HIAMP0438 gene, characterized in that, It is constructed with pHT43 as the backbone and contains the nucleotide sequence shown in SEQ ID NO.

4.

2. A recombinant Bacillus subtilis strain containing the black soldier fly antimicrobial peptide HIAMP0438 gene, characterized in that, The strain is Bacillus subtilis WB800N and contains the recombinant expression vector as described in claim 1.

3. The application of the recombinant expression vector as described in claim 1 or the recombinant Bacillus subtilis as described in claim 2 in the preparation of the black soldier fly antimicrobial peptide HIAMP0438.

4. The application according to claim 3, characterized in that, The amino acid sequence of the black soldier fly antimicrobial peptide HIAMP0438 is shown in SEQ ID NO.

1.

5. A method for preparing the black soldier fly antimicrobial peptide HIAMP0438, characterized in that, Using the recombinant Bacillus subtilis as described in claim 2, the black soldier fly antimicrobial peptide HIAMP0438 was produced under the induction of isopropyl-β-D-thiogalactoside.

6. The preparation method according to claim 5, characterized in that, The concentration of the isopropyl-β-D-thiogalactoside in the culture medium was 1 mmol / L.

7. The use of the recombinant Bacillus subtilis as described in claim 2 in the preparation of a drug for preventing and treating diseases caused by Gram-negative bacteria.

8. The application according to claim 7, characterized in that, The Gram-negative bacteria include Escherichia coli, Salmonella, and Listeria monocytogenes.

9. A drug for preventing and treating diseases caused by Gram-negative bacteria, characterized in that, Includes the recombinant Bacillus subtilis as described in claim 2.

Images