Use of silkworm silk gland BmSPI45 protein in preparation of anti-red turf fungus product

Abstract

The application discloses application of a silkworm silk gland BmSPI45 protein in preparation of a product resisting red hair moss, and relates to the technical field of bioengineering, and has the technical scheme as follows: the application of the silkworm silk gland BmSPI45 protein in preparation of the product resisting red hair moss, and the amino acid sequence of the silkworm silk gland BmSPI45 protein is SEQ ID NO:1.The application uses an insect baculovirus expression system to perform in-vitro expression on a BmSPI45 recombinant protein, and obtains a recombinant pFastBac-BmSPI45 eukaryotic expression vector.The constructed pFastBac-BmSPI45 eukaryotic expression vector is transformed into a DH10Bac competent cell, and a recombinant Bacmid is extracted and obtained, the recombinant Bacmid is transfected, and the BmSPI45 protein is massively expressed.The BmSPI45 recombinant protein is subjected to antibacterial function research, and the result shows that the protein has the effect of resisting red hair moss, thereby providing a new protein alternative for development of an antibacterial preparation.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically, to the application of the silkworm silk gland BmSPI45 protein in the preparation of products against *Trichophyton rubrum*. Background Technology

[0002] Trichophyton rubrum typically appears as granular or villous fungi and is one of the most common pathogenic fungi in clinical practice. It can infect the skin, hair, and nail plates, often causing tinea cruris, tinea manuum, tinea pedis, and onychomycosis. Currently, treatment for skin diseases caused by this fungus mainly involves antifungal agents, such as miconazole, ketoconazole, terbinafine, and ciclopirox olamine. However, these antifungal agents have certain side effects, and long-term use can lead to drug resistance.

[0003] The silkworm (Bombyx mori) is a representative insect of the order Lepidoptera and also a silk-producing insect with high economic value. The silk gland is the only organ in the silkworm capable of synthesizing and secreting silk proteins, making it crucial for its silk-producing function. Due to significant differences in morphology and function, the silk gland is divided into four parts: the posterior silk gland (PSG), the middle silk gland (MSG), the anterior silk gland (ASG), and the spinneret. Silkworm silk is mainly composed of fibroin and sericin. Fibroin is synthesized and secreted in PSG cells, including Fib-H, Fib-L, and P25 proteins; sericin is synthesized and secreted in MSG cells, with different segments of the MSG secreting different sericins.

[0004] Related studies have shown that in addition to abundant filamentin, silk also contains high levels of serine protease inhibitors, including BmSPI45 (…). Figure 1 These inhibitors showed significant inhibitory activity against proteinase K and trypsin. Sequence alignment of these protease inhibitors with previously reported homologous inhibitors revealed that BmSPI45 possesses multiple tandem TIL domains and different amino acid residues at the P1 active site, suggesting potentially broader inhibitory activity. However, research on BmSPI45 in the silkworm silk gland is limited to date, its specific function remains unclear, and there are no studies on its antifungal effects. Therefore, the inventors propose the application of the silkworm silk gland BmSPI45 protein in the preparation of products against *Trichophyton rubrum*. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the silkworm silk gland BmSPI45 protein in the preparation of anti-Trichogramma rubrum products, thereby solving the above-mentioned problems.

[0006] The above-mentioned technical objective of the present invention is achieved through the following technical solution: the application of silkworm silk gland BmSPI45 protein in the preparation of anti-red erythromycete products, wherein the amino acid sequence of silkworm silk gland BmSPI45 is SEQ ID NO:1.

[0007] The present invention is further configured such that the silkworm silk gland BmSPI45 protein is a recombinant protein.

[0008] The present invention is further configured such that the recombinant protein is expressed using a eukaryotic expression vector.

[0009] The present invention is further configured such that: the eukaryotic expression vector is a recombinant pFastBac-BmSPI45 eukaryotic expression vector.

[0010] The present invention is further configured such that the recombinant protein is the BmSPI45 recombinant protein expressed in vitro in an insect baculovirus expression system.

[0011] The present invention is further configured as follows: the method for obtaining the recombinant pFastBac-BmSPI45 eukaryotic expression vector is as follows: amplify the target fragment, separate it by electrophoresis, recover the target fragment for T cloning, sequence the PCR-positive single colonies of the bacterial culture, expand the sequenced single colonies, extract the plasmid, digest it with double enzymes, ligate the recovered target band with the expression vector pFastBac product recovered after double enzyme digestion, verify it by bacterial culture PCR, and then send it for sequencing to obtain the recombinant pFastBac-BmSPI45 eukaryotic expression vector.

[0012] The present invention is further configured such that the target fragments are BmSPI45-p10 and BmSPI45-pPH, whose serial numbers are SEQ ID NO:2 and SEQ ID NO:3, respectively.

[0013] The present invention is further configured such that the product is an antibacterial preparation.

[0014] In summary, the present invention has the following beneficial effects:

[0015] This invention provides the application of the silkworm silk gland BmSPI45 protein in the preparation of products against *Trichophyton rubrum*. The amino acid sequence of the silkworm silk gland BmSPI45 protein is SEQ ID NO:1. This invention utilizes an insect baculovirus expression system to express the recombinant BmSPI45 protein in vitro, obtaining the recombinant pFastBac-BmSPI45 eukaryotic expression vector. The constructed pFastBac-BmSPI45 eukaryotic expression vector is then transformed into DH10Bac competent cells, and recombinant Bacmid is extracted. Transfection with the recombinant Bacmid results in the large-scale expression of the BmSPI45 protein. The antibacterial function of the recombinant BmSPI45 protein was studied, and the results showed that this protein has an effect against *Trichophyton rubrum*, providing a new protein candidate for the development of antibacterial agents. Attached Figure Description

[0016] Figure 1 This invention relates to the application of the silkworm silk gland BmSPI45 protein in the preparation of an anti-Trichogramma rubrum product.

[0017] Figure 2 This describes the chromosomal location and gene structure of the silk gland BmSPI45 in the silkworm in this embodiment of the invention.

[0018] Figure 3 This is an amino acid sequence analysis of the silk gland BmSPI45 in the embodiment of the present invention;

[0019] Figure 4 This is the prokaryotic expression of BmSPI45 in the silk gland of silkworms in this embodiment of the invention. Figure 1 ;

[0020] Figure 5 This is the prokaryotic expression of BmSPI45 in the silk gland of silkworms in this embodiment of the invention. Figure 2 ;

[0021] Figure 6 This embodiment of the invention uses the pET-32a-BmSPI45 vector for prokaryotic expression.

[0022] Figure 7 This is a truncated BmSPI45 prokaryotic expression diagram in an embodiment of the present invention;

[0023] Figure 8 This is an example of the activity detection of truncated BmSPI45 recombinant protein in this invention.

[0024] Figure 9 This is the construction of the BmSPI45 eukaryotic expression vector in the embodiments of the present invention;

[0025] Figure 10 This refers to the construction and identification of recombinant Bacmid in this embodiment of the invention;

[0026] Figure 11 This is the antibacterial activity of the BmSPI45 recombinant protein in the embodiments of the present invention (the standard deviation is composed of 3 biological replicates).

[0027] Figure 12 This invention relates to the antibacterial activity of recombinant BmSPI45 protein against Trichophyton rubrum in the embodiments of the present invention (wherein, A: growth curve of recombinant BmSPI45 protein against Trichophyton rubrum; B: inhibitory effect of BmSPI45 on the growth of Trichophyton rubrum after 96 h of incubation; C: growth curve of BSA against Trichophyton rubrum; D: inhibitory effect of BSA on the growth of Trichophyton rubrum after 96 h of incubation; the relative standard deviation is composed of 3 biological replicates (*P<0.05)).

[0028] Figure 13 This invention presents the antibacterial activity of recombinant BmSPI45 protein against Beauveria bassiana in the embodiments of the present invention (A: growth curve of recombinant BmSPI45 protein against Beauveria bassiana; B: inhibitory effect of BmSPI45 on the growth of Beauveria bassiana after 96 h of incubation; C: growth curve of BSA against Beauveria bassiana; D: inhibitory effect of BSA on the growth of Beauveria bassiana after 96 h of incubation; the relative standard deviation is composed of 3 biological replicates). Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Experimental materials:

[0031] The silkworm strain D9L was preserved in our laboratory. After hatching, the silkworm eggs were fed fresh mulberry leaves. The incubation conditions were 25℃, relative humidity (75±5)%, and a photoperiod of 12L:12D. The silkworms were fed fresh mulberry leaves three times a day until they reached the cocooning stage. The pET-28a expression vector was preserved in our laboratory. BL21(DE3) and Trans1-T1 competent cells were purchased from Beijing TransGen Biotech Co., Ltd.

[0032] Experimental reagents and instruments

[0033] Table 1 Main experimental reagents

[0034]

[0035] Table 2 Main Experimental Instruments

[0036]

[0037] Example 1: BmSPI45 Bioinformatics Analysis

[0038] The gene and protein sequences of the silkworm BmSPI45 were downloaded from the NCBI database. The physicochemical properties of the protein were predicted and analyzed using the online software Protparam and ProtScale. The signal peptide was predicted using the online software SignaIP 6.0. Homology comparison was performed using the NCBI database, and the corresponding amino acid sequences were downloaded. A phylogenetic tree was constructed using MEGA 11.0 software.

[0039] The gene sequence downloaded from NCBI (Gene ID: 101739956) was compared with SilkDB 3.0 and it was found that the BmSPI45 gene is located on chromosome 6 of the silkworm and has 8 exons and 7 introns. Figure 2 Its CDS consists of 1184 bases, encoding 394 amino acids. Using the online software SignaIP 6.0, it was predicted that the protein has a 13-amino acid signal peptide, indicating that BmSPI45 is a secreted protein. Figure 3 Furthermore, the molecular weight and isoelectric point of the BmSPI45 protein were predicted. As shown in Table 1, the mature BmSPI45 protein has a molecular weight of 41.7 kDa, an isoelectric point of 4.61, and three tandem TIL domains containing 10 cysteine ​​residues each. Figure 3 ).

[0040] Table 2. Sequence Feature Analysis of BmSPI45

[0041]

[0042] Example 2: RNA extraction and reverse transcription

[0043] 2.1 RNA extraction

[0044] All experiments were conducted on ice at low temperatures, and all centrifugation conditions were performed at a low temperature of 4°C.

[0045] (1) Clean the dissection tools thoroughly, sterilize them under high pressure and dry them for later use.

[0046] (2) Take the silk glands, head, midgut, gonads, fat body, Malpighian tubules, epidermis, and the posterior silk glands of D9L larvae from the 5th instar to the 1st day of the 4th instar molting period into an RNase-free centrifuge tube and add 300 μL of Trizol solution.

[0047] (3) After grinding the tissue material thoroughly with a grinding rod, add 700 μL of Trizol solution, vortex the mixture thoroughly, and let it stand on ice for 10 min.

[0048] (4) Centrifuge at 13,000 rpm for 10 min in a low-temperature high-speed centrifuge.

[0049] (5) Do not aspirate the precipitate. Carefully transfer the supernatant to a new centrifuge tube, add one-fifth of the volume of Trizol solution in chloroform, shake until the mixture turns milky white, and let stand on ice for 10 min.

[0050] (6) Centrifuge at 13,000 rpm for 10 min in a low-temperature high-speed centrifuge. Remove the centrifuge tube and carefully transfer the top layer of solution into a new centrifuge tube. Do not transfer the second layer of solution.

[0051] (7) Add half the volume of Trizol solution to the above solution, mix thoroughly, and let stand for 10 minutes.

[0052] (8) Centrifuge at 13,000 rpm for 10 min in a low-temperature high-speed centrifuge. Carefully discard the supernatant, add an equal volume of 75% ethanol to the Trizol solution, and gently invert the centrifuge.

[0053] (9) Centrifuge at 8000 rpm for 5 min in a low-temperature high-speed centrifuge, discard the supernatant, carefully aspirate the remaining liquid at the bottom of the tube, open the tube cap, and dry the RNA.

[0054] (10) Dissolve the precipitate in an appropriate amount of NaAc-SDS solution, detect the integrity of the RNA, and determine its concentration and purity.

[0055] (11) Purify RNA: First, add 80 μL of RNase-free water to the centrifuge tube, then add anhydrous ethanol (2.5 times the volume) and 3 M sodium acetate (0.1 times the volume). Mix these solutions evenly and let them stand overnight or for 4 hours in a -20°C refrigerator.

[0056] (12) RNA repurification: Repeat steps 8-11 above and dissolve in RNase-free water.

[0057] 2.2 RNA reverse transcription

[0058] (1) Mix the reagents according to the table below, react at 70℃ for 5 min, immediately put them in an ice bath for ≥5 min, and then centrifuge.

[0059] Table 3 Reverse transcription system 1

[0060]

[0061] (2) Prepare the reverse transcription system according to the table below, and add the mixture to the reaction system in the previous step.

[0062] Table 4 Reverse Transcription System 2

[0063]

[0064] (3) React at 25℃ for 5 min, 42℃ for 60 min, 70℃ for 15 min, and 16℃ for ∞, and add an appropriate volume of 1×TE dilution to the centrifuge tube.

[0065] Example 3: Construction of nuclear expression vector

[0066] 3.1 Cloning primers were designed using Snapgene software. The target fragment was amplified using the silk gland cDNA obtained from the 5th day of the 5th instar of silkworms in Example 2 as a template. After amplification, nucleic acid electrophoresis was performed for detection.

[0067] Table 5 Primers for prokaryotic expression vectors

[0068]

[0069] Note: Underlined areas are enzyme cleavage sites.

[0070] Mix the above solutions thoroughly and place them in a PCR instrument. Follow the program as follows: 95℃ for 5 min, 95℃ for 10 s, 55℃ for 15 s, 72℃ for 55 s, 35 cycles, 72℃ for 7 min, and store at 16℃.

[0071] 3.2 Glue Recycling

[0072] Follow the instructions for the gel recovery kit.

[0073] 3.3 Connection

[0074] The recovered fragments were ligated into the pMD 19-T simple vector.

[0075] Table 6 Connection System

[0076]

[0077] Connect at 16℃ for 12 hours.

[0078] 3.4 Transformation

[0079] (1) Remove Trans1-T1 competent cells and place them on ice to thaw.

[0080] (2) Carefully add the ligation mixture to the melted competent cells and let stand for 30 min.

[0081] (3) Place the test tube containing the above mixed solution in a 42°C heat shock for 30 s, and immediately insert it into ice for 2 min after the shock.

[0082] (4) Add liquid culture medium to centrifuge tubes and incubate at 220 rpm for 1 h in a shaker at 37°C for recovery.

[0083] (5) Spread the bacterial solution on Amp + On the tablet.

[0084] (6) Incubate overnight at 37°C.

[0085] 3.5 Screening and sequencing of positive clones

[0086] Pick a single colony into Amp + In the culture medium, the cells were cultured on a shaker for 4-6 hours. After bacterial PCR verification, samples of the correct size were selected for sequencing verification.

[0087] 3.6 Plasmid extraction

[0088] Expand the bacterial culture after sequencing to obtain plasmids by following the kit instructions.

[0089] 3.7 Double enzyme digestion

[0090] Perform double enzyme digestion experiments on the plasmid and pET-28a vector obtained in the previous step according to the double enzyme digestion system in the table below.

[0091] Table 7 Double enzyme digestion system

[0092]

[0093] The product was analyzed using a 1.5% agarose gel, and the gel was recovered.

[0094] 3.8 Connection: The connection experiment was conducted at 16℃ for 12 h.

[0095] Table 8 Connection System

[0096]

[0097] 3.9 Conversion: The steps are the same as in step 3.4, but the resistance is changed to K. + .

[0098] 3.10 Same as step 3.5, perform bacterial culture PCR verification.

[0099] Purification of prokaryotic expressed proteins

[0100] 3.21 Conversion

[0101] Transform the sequenced recombinant plasmid into BL21(DE3) competent cells using the same method as above.

[0102] 3.22 Expanded cultivation

[0103] Pick a single colony and place it in K + In resistant LB medium, culture on a shaker for 4-6 hours. Inoculate the activated cells into 5 mL of K-containing medium. + Expand the culture in LB liquid medium until OD 600 It is approximately 0.4-0.6.

[0104] 3.23 Induced Expression

[0105] Three time conditions were set: 37℃, 220 rpm for 4 h; 25℃, 220 rpm for 12 h; and 16℃, 220 rpm for 20 h. Five IPTG induction concentrations were set: 0 mM, 0.1 mM, 0.5 mM, 1 mM, and 2 mM.

[0106] 3.24 Bacterial cell collection

[0107] The bacterial culture was centrifuged at 4°C, 7000 rpm for 10 min to completely remove the supernatant, and then the bacteria were resuspended.

[0108] 3.25 Ultrasonic fragmentation

[0109] (1) After three freeze-thaw cycles, the cells were ultrasonically broken up, with a breaking time of 5 min.

[0110] (2) Centrifuge at 13000 g for 15 min at 4℃, and collect the supernatant and precipitate respectively.

[0111] 3.26 Detection of low-level protein expression

[0112] (1) Sample preparation and loading: Prepare protein and loading buffer in a ratio of 4:1, boil at 95℃ for 10 min, load in a certain order, and electrophoresis.

[0113] (2) Place the gel in Coomassie Brilliant Blue staining solution and perform shake staining for 30 min.

[0114] (3) Add decolorizing solution to the shaker to decolorize.

[0115] Example 4: Construction of the recombinant pFastBac Dual vector

[0116] 4.1 PCR amplification

[0117] Cloning primers were designed using Snapgene software, and the target fragment was amplified using the silk gland cDNA obtained in section 3.2.2 on day 5 of the 5th instar of silkworms. Nucleic acid electrophoresis was performed after amplification.

[0118] Table 9 Primers for eukaryotic expression vectors

[0119]

[0120] Note: Underlined areas are enzyme cleavage sites.

[0121] 4.2 The subsequent steps are the same as steps 3.2-3.6 in Example 3.

[0122] 4.3 Double enzyme digestion

[0123] The pFastBac Dual plasmid vector and the extracted plasmid were subjected to double enzyme digestion, respectively.

[0124] Table 10 Double Enzyme Digestion

[0125]

[0126] The product was analyzed by agarose gel electrophoresis and then recovered from the gel.

[0127] 4.4 The recycled adhesive product was bonded at 16°C for 12 h.

[0128] Table 11 Enzyme digestion and ligation system

[0129]

[0130] 4.5 Convert the linker product to Trans1-T1, following the same steps as in step 3.4 of Example 3.

[0131] 4.6 Select a single colony for PCR verification, following the same steps as step 3.5 in Example 3.

[0132] Example 5: Construction and identification of recombinant Bacmid

[0133] 5.1 Take out 200 μL of DH10Bac competent cells and thaw them on ice.

[0134] 5.2 Add the recombinant pFastBac vector and the empty pFastBac vector, and incubate on ice for 30 min.

[0135] 5.3 Heat shock at 42℃ for 30 s, then immediately place on ice and let stand for 2 min.

[0136] 5.4 Add 500 μL of antibiotic-free LB medium and incubate at 37℃ and 220 rpm for 4 h.

[0137] 5.5 Remove the transformation solution, aspirate the bacterial culture and spread it on LB agar plates containing X-gal and IPTG triple antibodies, and incubate at 37°C for 36-48 h.

[0138] 5.6 After the blue-white spots are clearly distinguished, pick the white spots and streak them again on LB plates containing X-gal and IPTG triple antibodies, and incubate at 37°C for 36-48 h.

[0139] 5.7 Pick white spots and incubate them overnight at 37°C and 220 rpm in liquid LB medium containing triple antibodies. Then identify them by PCR.

[0140] 5.8 Collect positive bacteria and culture overnight. Centrifuge at 10,000 g for 1 min to collect the bacteria and discard the supernatant.

[0141] 5.9 Add 300 μL of Solution I (with added RNase A) to resuspend the bacterial pellet at the bottom of the tube.

[0142] 5.10 Add 300 μL of solution II and gently invert 4-6 times to completely lyse the bacteria.

[0143] 5.11 Add 300 μL of solution III and mix by inverting.

[0144] 5.12 Centrifuge at 12000 g for 10 min, and transfer the supernatant to a new centrifuge tube.

[0145] 5.13 Carefully add 800 μL of isopropanol to the centrifuge tube, mix gently and thoroughly, and let stand on ice for 10 min.

[0146] 5.14 Centrifuge at 13,000 rpm for 10 min in a low-temperature high-speed centrifuge and collect the precipitate.

[0147] 5.15 Add 500 μL of 70% ethanol to the centrifuge tube to resuspend the precipitate, centrifuge at the maximum speed of a low-temperature high-speed centrifuge for 5 min, and discard the supernatant.

[0148] 5.16 Add 200 μL of 70% ethanol to the centrifuge tube and proceed as before.

[0149] 5.17 Open the lid and dry, then dissolve Bacmid in the TE solution.

[0150] 5.18 The recombinant Bacmid obtained by extraction was identified by PCR.

[0151] Example 6: Obtaining Recombinant Baculovirus

[0152] 6.1 Seed passageable Sf9 cells in 6-well plates, transfect them with the target gene plasmid, and culture at 27°C for 3-4 days.

[0153] 6.2 Centrifuge at 500 g for 10 min, collect the supernatant and transfer it into a new EP tube. This is the P1 generation virus.

[0154] 6.3 Decking.

[0155] 6.4 Add 200 μL of P1 generation virus to the wells of the experimental group and incubate at 27℃ for 3-4 days.

[0156] 6.5 Centrifuge at 500 g for 10 min and collect the supernatant, which is the P2 generation virus.

[0157] 6.6 Add 200 μL of P2 generation virus to 50 mL of Sf9 cells.

[0158] 6.7 Incubate at 27℃ and 110 rpm for approximately 3 days.

[0159] 6.8 Centrifuge at 500 g for 10 min and collect the supernatant.

[0160] 6.9 Cells obtained by centrifugation were used for trial expression, followed by sonication, protein purification by nickel column chromatography, and detection by SDS-PAGE and Western Blot.

[0161] Example 7: Purification of eukaryotic expressed proteins

[0162] 7.1 Based on the results of small-scale induced expression, select the optimal conditions for large-scale induced expression.

[0163] 7.2 Centrifuge at 4℃, 7000 rpm for 20 min and collect all bacterial cells.

[0164] 7.3 Resuspend the bacterial cells, crush them under high pressure, and then centrifuge.

[0165] 7.4 Nickel column affinity chromatography

[0166] a. Equilibration: After washing the nickel column, add Buffer I to equilibrate the nickel column.

[0167] b. Sample loading: Slowly add the protein solution into the column.

[0168] c. Select a suitable buffer solution based on the experiment to elute extraneous proteins.

[0169] d. Prepare imidazole solutions of various concentrations and elute the target protein.

[0170] e. Collect the eluents of imidazole at various concentrations and test their concentration and purity.

[0171] Example 8: Desalination and Concentration

[0172] 8.1 The liquid in the dropper of the PD-10 desalting column was drained and rinsed three times with ultrapure water.

[0173] 8.2 Equilibrate the PD-10 column with buffer: Fill the column with equilibration buffer and repeat 4 times, using about 25 ml of equilibration buffer.

[0174] 8.3 Sample loading: Add the sample to the column and discard the flow-through liquid.

[0175] 8.4 Add 3.5 mL of 100 mM Tris-HCl to the top of the column for elution.

[0176] 8.5 Collect the eluent protein solution.

[0177] 8.6 After column chromatography, wash thoroughly and store the column in 20% ethanol at 4°C.

[0178] 8.7 Select the appropriate ultrafiltration tube, wash the concentrating membrane three times with Milli-Q water, add 100 mM Tris-HCl or PBS to wet and centrifuge at 5000 g for 5 min.

[0179] 8.8 Add the purified protein solution, centrifuge at 5000 g at 4℃ until 2 mL of liquid remains in the tube, discard the lower layer of liquid, gently pipette the protein off the filter membrane, and store at -80℃.

[0180] 8.9 Determine protein concentration, see steps 5-10 of 3.2.7.

[0181] Example 9: Inhibitor In-Gel Active Staining

[0182] 9.1 When performing active gel electrophoresis, prepare the protease solution in advance. After electrophoresis, place the gel in the prepared protease solution and incubate at 37°C and 45 rpm for 20 min in a shaker.

[0183] 9.2 The protein gel was washed with ultrapure water for 10 seconds each time, for a total of 3 washes, and then allowed to stand at room temperature for 20 minutes.

[0184] 9.3 Add staining solution (mixed with matrix solution) to the staining container and incubate at 37°C and 45 rpm for 20 min in a shaker.

[0185] 9.4 Carefully discard the staining solution in the staining container, add ultrapure water to stop the staining reaction, and clean the glue surface.

[0186] Example 10: Determination of antibacterial activity

[0187] 10.1 Bacterial activation: In a clean bench, Escherichia coli, Trichophyton rubrum and Beauveria bassiana were activated respectively.

[0188] 10.2 Selection of single clones and bacterial culture: Escherichia coli was cultured at 37°C and 220 rpm until the OD value reached 0.5. 600 The value was approximately 0.2; the fungi *Trichophyton rubrum* and *Beauveria bassiana* were diluted to OD using PDB medium. 600 The value is around 0.2.

[0189] 10.3 Detection: The following liquids were added to 96-well cell culture plates, with a total volume of 200 µL: 100 µL of PBS for the negative control group; 100 µL of 100 mM EDTA for the positive control group; and BmSPI45 protein sample for the experimental groups. The final concentrations of BmSPI45 protein in each system were 0.02 mg / mL, 0.1 mg / mL, and 0.34 mg / mL, respectively. If the volume was insufficient, PBS buffer was added to make up the difference. Each group was tested in triplicate. The *E. coli* group was cultured at 37℃ and 45 rpm, and the fungal group was cultured at 28℃ and 45 rpm.

[0190] 10.4 The OD values ​​of the bacterial and fungal groups were measured using an ultraviolet spectrophotometer. The Escherichia coli group was measured every 1 hour for 12 hours; the fungal group was measured every 12 hours for 96 hours.

[0191] 10.5 Based on the measured absorbance values, plot the antibacterial curve.

[0192] The above experimental results:

[0193] Prokaryotic expression, purification, and activity assay of BmSPI45 protein

[0194] This invention first uses a prokaryotic expression system for recombinant expression. First, the pET-28a-BmSPI45 prokaryotic expression vector (…) is constructed. Figure 4 A), transformed into BL21(DE3) competent cells, and after exploring low-level expression conditions, it was found that ( Figure 4 The recombinant BmSPI45 protein was expressed in inclusion bodies, but inclusion body proteins are inactive. To increase the possibility of expressing the recombinant BmSPI45 protein in the supernatant, pGEX-4T-1-BmSPI45 with a solubilization tag was subsequently constructed. Figure 4 DF), pET-32a-BmSPI45 ( Figure 5 AC), pCold-SUMO-BmSPI45 ( Figure 5 The three prokaryotic expression vectors (DF) were used for small-scale trials, but unfortunately, the recombinant proteins expressed by all three vectors existed in the form of inclusion bodies. (See appendix for the staining diagram of small-scale expression at 25℃).

[0195] Because BmSPI45 is rich in cysteine, Origami2 (DE3) competent cells were selected for experiments, as these cells are more conducive to disulfide bond formation. The pET-32a-BmSPI45 vector was transformed into Origami2 (DE3) competent cells, and the experiment was conducted under two temperatures (37℃ and 16℃) and four IPTG induction concentrations. Figure 6 (AB), no high expression of recombinant BmSPI45 protein was found in the supernatant.

[0196] A review of previous literature on the successful expression of active recombinant serine protease inhibitor proteins revealed that these proteins were all below 20 kDa, while the theoretical molecular weight of BmSPI45 is approximately 41.7 kDa. Therefore, a region containing one of the TIL domains of the BmSPI45 gene was extracted to construct the pET-28a-BmSPI45-cut prokaryotic expression vector. Figure 7 AD), the constructed prokaryotic expression vector was transformed into BL21(DE3) Escherichia coli. The effects of temperature and IPTG induction concentration were explored through low-level expression. Expression was induced for 20 h at a final concentration of 0.1 mM IPTG in a shaker at 16℃, and the results were detected by SDS-PAGE and Western Blot. Figure 7 E). The results showed that the truncated BmSPI45 recombinant protein could be expressed in the supernatant, and a large amount of the protein was eluted with 800 mM imidazole.

[0197] After desalting the purified recombinant truncated BmSPI45 protein, the results of reactive gel staining showed that the BmSPI45 protein did not have inhibitory activity against proteinase K. Figure 8 A). To eliminate the influence of protein quantity and protease substrate on the experimental results, the loading amount of recombinant protein was increased, and trypsin (A) was added. Figure 8 B) and subtilisin ( Figure 8 C) Two protease substrates were tested, and the results showed that the purified supernatant protein did not have inhibitory activity against these proteases.

[0198] Eukaryotic expression, purification, and activity assay of BmSPI45 protein

[0199] Since the prokaryotic expression system failed to produce active BmSPI45 protein, this study used an insect baculovirus expression system to express recombinant BmSPI45 protein in vitro. First, the target fragment (…) was amplified. Figure 9After separation by electrophoresis, the target fragment was recovered for T-cloning. PCR-positive single colonies were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The correctly sequenced single colonies were amplified, and plasmids were extracted and double-digested. The recovered target band was ligated with the expression vector pFastBac product, which had also been double-digested and recovered. After verification by PCR, the samples were sent for sequencing. Figure 9 CF), to obtain the recombinant pFastBac-BmSPI45 eukaryotic expression vector.

[0200] The constructed pFastBac-BmSPI45 eukaryotic expression vector was then transformed into DH10Bac competent cells, and after blue-white screening and bacterial PCR verification, ( Figure 9 (AB) The monoclonal white spot disease was cultured in a large scale, and recombinant Bacmid was extracted.

[0201] Recombinant Bacmid was transfected into Sf9 cells to obtain P1 generation recombinant baculovirus. The P1 generation virus was then further amplified to obtain P2 and P3 generation viruses. The obtained P3 generation virus was used to infect Sf9 cells for high-level expression of BmSPI45 protein. Experimental results are as follows: Figure 10 As shown in Figures A and B, SDS-PAGE analysis revealed that BmSPI45 protein was eluted with 100 mM imidazole. Western blotting showed that the protein band at this location successfully bound to the BmSPI45 antibody, indicating successful expression of the BmSPI45 protein. The purified recombinant BmSPI45 protein was then desalted and concentrated. Active gel staining results showed that the BmSPI45 protein exhibited strong inhibitory activity against proteinase K. Figure 10 C).

[0202] Assay of antibacterial activity of BmSPI45 protein

[0203] To detect the antibacterial activity of BmSPI45 protein, *E. coli* was co-incubated with different concentrations of BmSPI45 protein for 12 h, and the OD value was measured every 1 h to plot the growth curve of *E. coli*. Figure 11 It is evident that the BmSPI45 protein cannot inhibit the growth of Escherichia coli.

[0204] To determine whether BmSPI45 protein has inhibitory activity against fungi, *Trichophyton rubrum* and *Beauveria bassiana* were co-incubated with BmSPI45 protein at final concentrations of 0.02 mg / mL, 0.1 mg / mL, and 0.34 mg / mL for 96 h. OD values ​​were measured every 12 h to plot fungal growth curves. PBS and corresponding concentrations of BSA protein were used as negative controls, and EDTA as a positive control. The inhibitory activity of BmSPI45 protein against fungi was investigated. Figure 12In the inhibition experiment, the positive control results showed that EDTA had a significant inhibitory effect on the growth of Trichophyton rubrum; in the negative control experiment, PBS and BSA had no effect on the growth of Trichophyton rubrum. The BmSPI45 protein in the experimental group could inhibit the growth of Trichophyton rubrum. Figure 12 A), and this inhibitory effect increases with increasing BmSPI45 concentration. For Beauveria bassiana (… Figure 13 In the inhibition experiment, the positive control showed an inhibitory effect on the growth of *Beauveria bassiana*, while the negative control had no effect. Different concentrations of BmSPI45 did not significantly change the growth of *Beauveria bassiana* compared to the negative control, indicating that it has no inhibitory effect on *Beauveria bassiana*. These results indicate that BmSPI45 has a significant inhibitory effect on *Trichophyton rubrum*, but no inhibitory effect on *Beauveria bassiana*.

[0205] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of BmSPI45 protein from the silk gland of silkworms in the preparation of products against Trichophyton rubrum, characterized by: The amino acid sequence of the silkworm silk gland BmSPI45 protein is SEQ ID NO:

1.

2. The application according to claim 1, characterized in that: The silk gland BmSPI45 protein in silkworms is a recombinant protein.

3. The application according to claim 2, characterized in that: The recombinant protein was expressed using a eukaryotic expression vector.

4. The application according to claim 3, characterized in that: The eukaryotic expression vector is the recombinant pFastBac-BmSPI45 eukaryotic expression vector.

5. The application according to claim 2, characterized in that: The recombinant protein is the BmSPI45 recombinant protein expressed in vitro in an insect baculovirus expression system.

6. The application according to claim 4, characterized in that: The steps for obtaining the recombinant pFastBac-BmSPI45 eukaryotic expression vector are as follows: amplify the target fragment, separate it by electrophoresis, recover the target fragment for T cloning, sequence the PCR-positive single colonies of the bacterial culture, expand the sequenced single colonies, extract the plasmid, digest it with two enzymes, ligate the recovered target band with the expression vector pFastBac product recovered after double enzyme digestion, verify it by bacterial culture PCR, and then send it for sequencing to obtain the recombinant pFastBac-BmSPI45 eukaryotic expression vector.

7. The application according to claim 6, characterized in that: The target fragments are BmSPI45-p10 and BmSPI45-pPH, with serial numbers SEQ ID NO:2 and SEQ ID NO:3, respectively.

8. The application according to claim 1, characterized in that: The product is an antibacterial agent.

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