Targeting material for microorganisms and application thereof

Abstract

The invention discloses a microbial targeting material and application thereof. The microbial targeting material provided by the invention comprises a solid-phase carrier and phage cell wall binding domain protein crosslinked on the solid-phase carrier. According to the microbial targeting material disclosed by the invention, the phage cell wall binding domain protein is creatively combined with the solid-phase carrier, target microorganisms are specifically identified and bound by utilizing the phage cell wall binding domain protein, and then the target microorganisms are separated and screened by utilizing the solid-phase carrier; a rapid, efficient and high-throughput screening scheme and way are provided for screening of functional microorganisms; target microorganism screening based on the microorganism targeting material has the advantages of being high in sensitivity, good in specificity, high in acid and alkali resistance and the like, has good temperature adaptability and salt concentration adaptability, and further widens the application range of functional microorganisms.

Description

Technical Field

[0001] The present application relates to the technical field of functional microorganism screening, and in particular to a microorganism targeting material and its application. Background Art

[0002] Due to the diversity of microbial functions, they are widely used in medicine, industrial manufacturing, pesticide production, food industry, environmental pollution remediation, and bioleaching of high-value minerals. Microbial genes also exhibit diverse functions and have been successfully applied in many fields such as biopharmaceuticals, agriculture, and environmental protection. Therefore, screening microbial strains with new functions or containing new functional genes has received great attention. Targeted screening of functional microorganisms from the environment is the key to microbial applications. At present, the screening of functional microorganisms mainly relies on the strategy of separation one by one, and there are few strategies for directly screening functional microorganisms through high-throughput methods.

[0003] Bacillus thuringiensis (Bt) is a widely distributed microorganism primarily used in the production of biopesticides. Currently, over 70% of commercial pesticides are derived from this bacterium. The insecticidal crystal proteins produced by B. thuringiensis have been successfully expressed in transgenic plants, conferring inherent insect resistance and driving significant progress in agriculture. Traditional methods for isolating B. thuringiensis primarily rely on antibiotic selectivity screening, sodium acetate selection screening, temperature screening, and physiological, biochemical, and serotype identification. These methods are time-consuming, subject to high inaccuracies, cumbersome procedures, and lack of sensitivity, making the isolation of new strains more difficult. Discovering parasporal crystal protein genes with novel activities is a potential strategy for expanding its application. The highly effective insecticidal activity of B. thuringiensis depends not only on its crystal proteins but also on the bacteria itself. Therefore, there is an urgent need to develop faster and more efficient techniques for screening new B. thuringiensis strains. Summary of the Invention

[0004] The purpose of this application is to provide a new microbial targeting material and its application.

[0005] This application adopts the following technical solutions:

[0006] The first aspect of the present application discloses a microorganism targeting material, comprising a solid phase carrier and a bacteriophage cell wall binding domain protein cross-linked on the solid phase carrier.

[0007] It should be noted that the present application creatively combines the phage cell wall binding domain protein with a solid phase carrier, such as magnetic beads, and utilizes the phage cell wall binding domain to specifically recognize and bind to target microorganisms to achieve their separation and screening.

[0008] In one implementation of the present application, the phage cell wall binding domain protein is the cell wall binding domain protein of the lytic enzyme PlyBt33 of Bacillus thuringiensis phage BtCS33.

[0009] In one implementation of the present application, the cell wall binding domain protein of the lytic enzyme PlyBt33 is the sequence shown in SEQ ID NO.1.

[0010] SEQ ID NO.1:

[0011] NKSYKQEGVEIIVNKHNKVITYEFGVNLIPEMIQMMDTLGYTSKIVSRGD RQGLVYFESDYRQGSELDKATAWLDAKGLKYFYTKE.

[0012] It should be noted that in one implementation of the present application, the cell wall binding domain of Bacillus thuringiensis phage was used as an example for experiments, which verified that the targeting material of the present application can indeed achieve the separation and screening of Bacillus thuringiensis. It can be understood that, first, the inventive concept of the present application of cross-linking the solid phase carrier with the phage cell wall binding domain protein to achieve the corresponding target microorganism separation and screening is not limited to Bacillus thuringiensis. For other strains, it is only necessary to replace the cell wall binding domain protein of the corresponding target microorganism phage; second, the present application specifically uses the cell wall binding domain protein of the lytic enzyme PlyBt33 of Bacillus thuringiensis phage BtCS33. Under the same inventive concept, it is not ruled out that the cell wall binding domain protein of the lytic enzyme of other Bacillus thuringiensis phages can also be used.

[0013] In one implementation of the present application, the solid phase carrier is a magnetic bead, a chip, a porous separation column or a metal nanoparticle.

[0014] In one implementation of the present application, the solid phase carrier has a modification group that cross-links with the protein, or a group with a metal ion is introduced into the solid phase carrier, and the metal ion is used to form a specific bond with a recombinant protein containing a histidine tag (His-tag).

[0015] In one implementation of the present application, the metal ions include Ni 2+ 、Co 2+ .

[0016] In one implementation of the present application, the modifying group includes a hydroxyl group.

[0017] It should be noted that the key to this application lies in the creative use of bacteriophage cell wall binding domain proteins to separate and screen target microorganisms. The main function of the solid phase carrier is fixation and separation. In principle, any solid phase carrier that can play this role can be used in this application, including but not limited to magnetic beads, chips or metal nanoparticles.

[0018] Another aspect of the present application discloses a recombinant plasmid comprising a gene fragment expressing a cell wall binding domain protein of the lytic enzyme PlyBt33 of Bacillus thuringiensis phage BtCS33.

[0019] Among them, the cell wall binding domain protein of the lytic enzyme PlyBt33 expressed by Bacillus thuringiensis phage BtCS33 is the sequence shown in SEQ ID NO.1.

[0020] In one implementation of the present application, the gene fragment of the cell wall binding domain protein of the lytic enzyme PlyBt33 is the sequence shown in SEQ ID NO.2.

[0021] SEQ ID NO.2:

[0022] AATAAATCGTATAAACAAGAAGGAGTGGAGATTATCGTGAACAAACATAATAAGGTGATTACTTATGAATTTGGTGTAAATTTAATTCCAGAAATGATTCAAATGATGGATACGCTTGGATACACTTCAAA AATTGTTTCTCGTGGAGATCGTCAGGGGCTTGTTTATTTTGAGTCGGATTATCGTCAAGGTAGCGAGCTAGATAAAGCAACAGCGTGGTTAGATGCTAAAGGACTTAAATACTTTTATACAAAAGAATAG.

[0023] Another aspect of the present application discloses a recombinant cell containing the recombinant plasmid of the present application.

[0024] It should be noted that the recombinant plasmids and recombinant cells of the present application can be used to prepare the cell wall binding domain protein of the lytic enzyme PlyBt33 of the Bacillus thuringiensis phage BtCS33. Further, the cell wall binding domain protein is cross-linked with a solid phase support to obtain the targeting material of the present application. Therefore, the recombinant plasmids and recombinant cells of the present application can be used to prepare the targeting material of the present application.

[0025] Another aspect of the present application discloses the use of the targeting material, the recombinant plasmid or the recombinant cell of the present application in the screening of Bacillus thuringiensis.

[0026] Another aspect of the present application discloses a kit for screening Bacillus thuringiensis, which comprises at least one of the targeting material of the present application, the recombinant plasmid of the present application, and the recombinant cell of the present application.

[0027] It can be understood that if the kit only contains the recombinant plasmid or the recombinant cell of the present application, it is necessary to prepare the cell wall binding domain protein of the lytic enzyme PlyBt33 of the Bacillus thuringiensis phage BtCS33, and then cross-link it with the solid phase support to obtain the targeting material of the present application before it can be used for the separation and screening of Bacillus thuringiensis.

[0028] Another aspect of the present application discloses a method for screening microorganisms, comprising contacting the targeting material of the present application with a sample, and screening the target microorganism from the sample through the binding of the targeting material and the target microorganism; the phage cell wall binding domain protein cross-linked on the solid phase carrier in the targeting material is provided by the phage of the target microorganism.

[0029] The beneficial effects of this application are:

[0030] The microbial targeting material of the present application creatively combines the phage cell wall binding domain protein with a solid phase carrier, uses the phage cell wall binding domain protein to specifically identify and bind to the target microorganism, and then uses the solid phase carrier to separate and screen the target microorganism, providing a rapid, efficient, and high-throughput screening scheme and approach for the screening of functional microorganisms; the target microorganism screening based on the microbial targeting material of the present application has the advantages of high sensitivity, good specificity, strong acid and alkali resistance, and has good temperature adaptability and salt concentration adaptability, further broadening the application range of functional microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the principle of screening target microorganisms using microbial targeting materials in the embodiments of the present application;

[0032] Figure 2 This is a diagram showing the characterization results of proteins PlyBt33-IC and MB-CWBD in the examples of this application;

[0033] Figure 3 This is a graph showing the test results of the binding ability of MB-CWBD to Bt strains in the examples of this application;

[0034] Figure 4 This is a graph showing the detection results of the binding selectivity of MB-CWBD to Bacillus thuringiensis strains in the examples of the present application;

[0035] Figure 5This is a graph showing the detection results of MB-CWBD binding to Bt strains from natural environment soil in the examples of this application. DETAILED DESCRIPTION

[0036] Bacteriophages are viruses that infect bacteria, and the lytic enzymes they encode are considered promising antibacterial substances due to their specificity and high lytic activity against bacterial cells. Most gram-positive bacterial phage lytic enzymes have a "dual domain" feature, with a catalytic domain (CD) at the N-terminus and a cell wall binding domain (CWBD) at the C-terminus. The cell wall binding domain (CWBD) has the ability to specifically recognize bacterial cells. Lytic enzymes have attracted much attention in the rapid detection of bacteria, especially pathogenic bacteria, and have shown potential application prospects in the fields of medicine, biotechnology, agriculture, and food safety. Although phage lytic enzymes are widely used, there are currently no literature reports on the application of the phage lytic enzyme cell wall binding domain combined with magnetic bead technology in the practice or research of functional microbial screening.

[0037] The surface of carboxyl magnetic beads is modified with abundant carboxyl groups and is widely used in protein purification, cell separation and molecular detection. Magnetic bead technology uses a magnetic field to achieve rapid separation of target antigens, improves detection specificity, significantly shortens detection time and enhances detection sensitivity. This study proposes to combine the cell wall binding domain of phage lytic enzyme with magnetic bead technology. By constructing a CWBD encoded by Bacillus thuringiensis phage lytic enzyme PlyBt33 on the surface of magnetic beads, a strategy for efficient screening of Bacillus thuringiensis strains was established. Figure 1 As shown in Figure 2, the modified magnetic beads MB-CWBD utilize CWBD's specific recognition ability for bacterial cells to target Bacillus thuringiensis. MB-CWBD exhibits highly stable and high-throughput binding to Bacillus thuringiensis from diverse environments. Given the diversity of phage lytic enzymes CWBD, the technology proposed in this study can also be used to screen for functional bacteria in other areas.

[0038] The present application is further described in detail below through specific examples. The following examples are only used to further illustrate the present application and should not be understood as limiting the present application.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0040] Example

[0041] 1. Materials and Methods

[0042] 1.1 Experimental Materials

[0043] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-(N-Morpholino)ethanesulfonic acid hydrate (MES), manganese sulfate (MnSO4), and imidazole were purchased from Macklin (Shanghai Macklin Biochemical Technology Co., Ltd.). Magnetic beads with particle sizes of 1 μm, 3 μm, and 5 μm were purchased from Xfnano (carboxylated magnetic microspheres, Xianfeng Nano, Jiangsu Xianfeng Nanomaterials Technology Co., Ltd.). Bovine serum albumin (BSA) was purchased from Sigma (Sigma-Aldrich (Shanghai) Trading Co., Ltd.). The carboxyl magnetic bead coupling kit and N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) were purchased from Sangon (Shanghai Shenggong Biotechnology Co., Ltd.). Coomassie Brilliant Blue R-250 was purchased from Solebol.

[0044] 1.2 Characterization methods

[0045] The expressed and purified proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was determined using a microplate reader (Multiskan SkyHigh with Touch Screen, Thermo Scientific, USA). The microstructure of the magnetic beads was observed using a scanning electron microscope (SEM, 5 kV, Hitachi SU8020, Japan). The infrared spectra of the magnetic beads were observed using a Fourier transform infrared spectrometer (FTIR) (Nicolet IS10, ThermoScientific, USA). Circular dichroism (CD) spectroscopy (J-715, JASCO, Tokyo, Japan) was used to detect changes in the secondary structure of the purified protein before and after cross-linking with the magnetic beads. The bacterial spores and paraspore crystals were observed using an optical microscope (BA210, Motic, USA) and a scanning electron microscope.

[0046] 1.3 Preparation of bacteriophage lytic enzyme CWBD protein

[0047] Sangon Biotech (Shanghai) Co., Ltd. artificially synthesized the CWBD protein gene fragment, PlyBt33-IC. This gene fragment was inserted into the BamHI / HindIII cloning sites of the pET28a(+) vector, and the recombinant plasmid pET28a-PlyBt33-IC was transformed into Escherichia coli BL-21 to construct the recombinant strain E. coli BL-21pET28a-PlyBt33-IC. IPTG was added to a final concentration of 1 mM, and the cells were induced at 16°C for 8 h. The CWBD protein was expressed in the recombinant strain and purified by nickel-chelate affinity chromatography. Protein concentration was determined by the Bradford method. The purified protein was further dialyzed against crosslinking buffer (MES, 0.05 M, pH 6.5) for subsequent studies.

[0048] 1.4 Construction of magnetic beads displaying CWBD

[0049] To construct magnetic beads displaying the cell wall binding domain of the bacteriophage lytic enzyme PlyBt33, purified CWBD protein was covalently cross-linked to carboxyl magnetic beads. The carboxyl magnetic beads were first activated, washed three times with coupling buffer, and then suspended in coupling buffer to a final concentration of 0.5 mg mL -1 Add a final concentration of 0.25 mg mL -1 of EDC and the final concentration was 0.25 mg mL -1 NHS was added to a final concentration of 0.15 mg mL -1 The CWBD protein solution was added to the activated magnetic beads and mixed evenly. The mixture was placed on a rotator and incubated at room temperature for 90 min to obtain cross-linked magnetic beads MB-CWBD. The cross-linked magnetic beads MB-CWBD were incubated with PBS (0.24 g L - 1 KH2PO4, 1.44 g L -1 Na2HPO4, 8g L -1 NaCl, 0.2 g L -1 The cells were washed three times with KCl (pH 7.4) and used for subsequent studies. BSA was cross-linked with carboxyl magnetic beads using the same method as above to obtain magnetic beads MB-BSA, which served as a control group for subsequent studies. The final concentrations were 0.01 mg mL -1 , 0.02mg mL -1 , 0.04mg mL -1 , 0.06mg mL -1 , 0.08mg mL -1 , 0.10mg mL -1 , 0.15mg mL -1 , 0.20mg mL -1 , 0.40mg mL -1, 0.60mg mL -1 The CWBD protein was added to a final concentration of 0.5 mg mL -1 The cross-linking rate of the beads with different protein concentrations was analyzed by cross-linking the carboxyl groups of the beads. The cross-linking rate was calculated using formula (1). SEM micrographs of the beads before and after cross-linking with BSA and CWBD proteins were observed. FTIR was used to detect differences in the beads before and after cross-linking with CWBD protein. CD was used to determine changes in the secondary structure of the protein before and after cross-linking with the beads.

[0050] Formula (1) Cross linking rate = (C pini -C pres )÷C pini ×100%

[0051] Among them, C pini and C pres represent the initial protein concentration and the residual protein concentration, respectively.

[0052] Binding performance of 1.5MB-CWBD to Bacillus thuringiensis

[0053] The binding ability of MB-CWBD to Bacillus thuringiensis BMB171 was tested using the indicator bacterium. Exponentially growing strain BMB171 was harvested by centrifugation, washed three times with PBS, and resuspended in PBS. The turbidity of the suspension was assessed by measuring absorbance at 600 nm.

[0054] MB-CWBD was added to the BMB171 bacterial suspension (final concentration was 4.2×10 7 CFU mL -1 ) to a final concentration of 0.5 mg mL -1 , shake for 1 h, and measure the turbidity of the bacterial solution after removing the magnetic beads to evaluate the residual bacterial concentration after adsorption.

[0055] The binding performance of MB-BSA and MB to Bacillus thuringiensis BMB171 was determined by the above method. The final concentration was 0.0375 mg mL -1 , 0.0625mg mL -1 , 0.125mg mL -1 , 0.25mg mL -1 , 0.5mg mL -1 , 1mg mL -1 MB-CWBD of different concentrations were added to the BMB171 bacterial suspension to compare the differences in the binding performance of MB-CWBD with Bacillus thuringiensis.

[0056] The final concentration was 0.5 mg mL -1MB-CWBD was added to the bacterial suspension of Bacillus thuringiensis BMB171 and cultured with shaking. The concentration of the remaining bacterial solution was measured every 10 minutes to determine the optimal adsorption time of MB-CWBD on Bacillus thuringiensis. MB-BSA was used as a control.

[0057] The effect of pH on the binding capacity of MB-CWBD was determined by adjusting the pH of the bacterial solution to pH 1.0-pH 11.0.

[0058] The effect of temperature on the binding properties of MB-CWBD was determined by setting different temperatures of 5°C, 25°C, 28°C, 35°C, 40°C, and 50°C.

[0059] The effect of salt concentration on the binding properties of MB-CWBD was achieved by setting different NaCl solution concentrations of 0 mM, 50 mM, 100 mM, 150 mM, 200 mM, and 250 mM.

[0060] The final concentration was 0.5 mg mL -1 MB-CWBD were added into bacterial suspensions with different pH, temperature and salt concentration.

[0061] The effect of magnetic beads with different particle sizes on the binding ability of MB-CWBD was studied by adding MB-CWBD constructed with magnetic beads with particle sizes of 1 μm, 3 μm, and 5 μm to the bacterial suspension to a final concentration of 0.5 mg mL -1 , 1mg mL -1 , 2mg mL -1 , 3mg mL -1 , 4mgmL -1 To be determined.

[0062] The binding rate was calculated using formula (2).

[0063] Formula (2) Capturing rate = (C ini -C res )÷C ini ×100%

[0064] Among them, C ini and C res represent the concentrations of the initial bacterial suspension and the residual bacterial suspension, respectively.

[0065] Determination of the binding selectivity of 1.6MB-CWBD to different strains

[0066] In order to determine the binding selectivity of MB-CWBD to different bacterial strains, the strains to be tested (as shown in Table 1) at different exponential growth phases were collected by centrifugation and washed three times with PBS. The bacterial cells were resuspended in PBS to an OD of600nm MB-CWBD was added to the bacterial suspension to a final concentration of 0.5 mg mL -1 MB-CWBD was removed by magnetic separation and the concentration of the residual bacterial suspension was determined. The binding rates of MB-CWBD to different strains were calculated according to the above formula (2) and the differences were compared.

[0067] Table 1 Test strains

[0068]

[0069]

[0070] 1.7 Determination of the selectivity of CWBD and MB-CWBD for binding of Bacillus thuringiensis spores to vegetative cells

[0071] The spore and vegetative suspensions of Bacillus thuringiensis were prepared to test the binding performance of CWBD and MB-CWBD on their spores and vegetative bodies. -1 NaCl, 10gL -1 Peptone, 5 g L -1 Yeast powder, 15g L -1 Agar powder, 0.06 g L -1 MnSO4), eluted with PBS and the spores were collected and resuspended. The above suspension was shaken at low temperature to fully break up the spores and vegetative cells, centrifuged and the supernatant was discarded, and the precipitate was washed 5 times to remove the vegetative cells. The spores were resuspended in ACES buffer (N-(2-acetamido)-2-aminoethanesulfonic acid, 0.05M, pH 7.0) and treated at 80°C for 20 minutes to completely remove the vegetative cells. The vegetative suspension was prepared and the exponential growth phase strain BMB171 was inoculated into LB liquid medium (10 g L -1 NaCl, 10 g L - 1 peptone, 5g L -1 Yeast powder) and cultured at 25°C and 180 rpm for 5 h. Collect the precipitate and resuspend it in PBS buffer. Add the spore suspension and vegetative suspension to CWBD protein (final concentration of 0.37 mg mL -1 ) to a final concentration of 4.05×10 6 CFU mL -1 and 4.07×10 6 CFU mL -1Samples were taken every 10 minutes to determine the protein concentration remaining in the liquid after adsorption of spores and vegetative bodies. The changes in protein concentration during the binding of BSA to spores and vegetative bodies were determined using the same method as above. In order to determine the binding performance of MB-CWBD to spores and vegetative bodies, the constructed MB-CWBD was added to the above spore suspension (final concentration of 4.05×10 7 CFU mL -1 ), vegetative suspension (final concentration of 4.07×10 7 CFU mL -1 ) to a final concentration of 0.5 mg mL -1 Oscillate at moderate speed for 1 hour. Remove MB-CWBD by magnetic separation and determine the concentration of the residual bacterial suspension. Calculate the binding rate of MB-CWBD to spores and vegetative cells according to the above formula (2) and compare the differences. Use MB-BSA to determine the binding rate of MB-CWBD to spores and vegetative cells as a control.

[0072] Binding performance of 1.8MB-CWBD to Bacillus thuringiensis in a simulated natural environment

[0073] In order to determine the binding performance of MB-CWBD to Bacillus thuringiensis in a simulated natural soil environment, soil samples collected from the natural environment were first sterilized with high pressure steam for 1 hour to fully kill the bacteria in the soil. Bacillus thuringiensis BMB171 in the exponential growth phase was added to the soil and stirred evenly. 1 g of soil was further resuspended in 9 mL of sterile water to a final bacterial suspension concentration of 10 7 CFU mL -1 MB-CWBD was added to the soil suspension at a final concentration of 0.5 mg mL -1 After 1 h of interaction, MB-CWBD was removed by magnetic separation and the concentration of the residual bacterial suspension was determined. The binding rate of MB-CWBD to Bacillus thuringiensis was calculated according to the above formula (2).

[0074] Binding performance of 1.9MB-CWBD against different strains in a simulated natural environment

[0075] In order to determine the binding differences of MB-CWBD to different strains in a simulated natural soil environment, the same bacterial solution concentration (bacterial solution concentration of 10 7 CFU mL -1 ) were added to the soil. MB-CWBD was added to the soil suspension at a final concentration of 0.5 mg mL -1After 1 h of interaction, MB-CWBD was removed by magnetic separation and the concentration of the residual bacterial suspension was determined. The binding rates of MB-CWBD to different strains were calculated according to the above formula (2) and their differences were compared.

[0076] In addition, to examine the differences in the binding ability of MB-CWBD to closely related strains of Bacillus thuringiensis, Bacillus thuringiensis BMB171, Bacillus subtilis CMCC 63501G3, Staphylococcus aureus ATCC 6538G3, and Escherichia coli CMCC 40482 were added to the soil using the above method. Samples were taken every 10 minutes, and the MB-CWBD was removed by magnetic separation. The concentration of the residual bacterial suspension was measured. The binding rates of MB-CWBD to the different strains were calculated according to the above formula (2) and the differences were compared.

[0077] Binding performance of 10MB-CWBD to Bacillus thuringiensis in natural environment

[0078] Soil samples were collected from the campus of Hainan Normal University in Haikou, Hainan Province (110°20′E, 19°59′N) to test the ability of MB-CWBD to isolate Bacillus thuringiensis from natural environments. Soil samples were collected at a depth of 5 cm using a five-point sampling method. 2 g of soil sample was collected from each point. The soil samples from the five points were cleaned of impurities, ground and mixed, and 10 g of soil sample was resuspended in 90 mL of sterile water to a final concentration of 0.1 g mL. -1 The soil suspension was placed at 37°C and shaken at 180 rpm for 1 h, then filtered through gauze. The supernatant was collected and MB-CWBD was added to the soil suspension to a final concentration of 0.5 mg mL -1 After 1 h of interaction, MB-CWBD was collected by magnetic separation.

[0079] MB-CWBD was further resuspended in 100 μL of blocking solution, and the residual bacterial suspension was collected. The soil suspension, blocking suspension, and residual bacterial suspension were gradiently diluted and spread respectively, cultured for 5 days to observe the colony morphology, and the suspected colonies were stained by alkaline fuchsin staining and observed by optical microscopy. The bacteria were collected and fixed with 2.5% glutaraldehyde fixative at 4°C in the dark for 24 hours. After dehydration, they were critically dried, sprayed with gold, and then observed by scanning electron microscopy. The strains that were observed to produce spores and paraspore crystals were preliminarily identified as Bacillus thuringiensis strains. The traditional Bacillus temperature separation method was used as a control. The soil suspension was boiled in water for 10 minutes and then gradiently diluted and spread for observation. The experimental results were recorded and the differences between different separation strategies were compared.

[0080] 2. Results and Discussion

[0081] Characterization of 2.1MB-CWBD

[0082] The magnetic beads MB-CWBD displaying the cell wall binding domain of bacteriophage lytic enzyme PlyBt33 were successfully prepared by covalent cross-linking method. Figure 1 As shown. The size of PlyBt33-IC protein is 11kDa. Figure 2 As shown in Figure a, the amino group at its N-terminus is responsible for cross-linking with the carboxyl groups on the surface of the magnetic beads. The binding mechanism between magnetic beads and proteins is generally covalent, and the stability of the binding is enhanced by charge interactions. The results show that the concentration at which the protein cross-linking rate on the magnetic beads is the highest is 31.66 μg mg -1 ,like Figure 2 As shown in Figure b. Based on the protein concentration in MB-CWBD and the molecular weight of PlyBt33-IC protein, it was calculated that the maximum binding capacity of MB-CWBD for each binding module without considering steric hindrance is 1.73×10 15 CFU mg -1 (Capturemax=C pmax ÷MW×N A , where C pmax represents the maximum concentration of protein cross-linking, MW represents the relative molecular weight of protein, N A Represents Avogadro's constant), has a strong cross-linking ability, enough to bind a large number of BT cells, the magnetic beads were named MB-CWBD for subsequent research. SEM observation of the magnetic beads showed that during the cross-linking process, the magnetic beads showed an integrated structure, such as Figure 2 As shown in Figure c. FTIR confirmed that the protein was successfully cross-linked with the magnetic beads. After cross-linking with the protein, the C═O peak, amide bond (-CO-NH-) peak, and NH peak of the protein component were observed, as shown in Figure c. Figure 2 As shown in Figure d. The protein before and after cross-linking with magnetic beads was detected by circular dichroism, with a detection wavelength range of 190 to 260 nm and a bandwidth of 1 nm. The positive absorption peak at 200 nm is the characteristic peak of circular dichroism of β-sheet structure. The secondary structure is mainly antiparallel β-sheet. The secondary structure of the protein did not change significantly after cross-linking with magnetic beads, as shown in Figure d. Figure 2 As shown in Figure e.

[0083] Figure 2 Figures 2 and 3 are the characterization results of proteins PlyBt33-IC and MB-CWBD, where (a) is the SDS-PAGE gel electrophoresis of the purified proteins PlyBt33-IC and BSA; (b) is the concentration of the cross-linked protein PlyBt33-IC on the magnetic beads; (c) is the SEM image of MB, MB-BSA, and MB-CWBD; (d) is the FTIR spectrum of MB, MB-BSA, and MB-CWBD; and (e) is the circular dichroism spectrum of CWBD and MB-CWBD.

[0084] 2.2 Bt binding performance of MB-CWBD

[0085] The binding performance of magnetic beads with different particle sizes to Bacillus thuringiensis was further determined. Figure 3 As shown in a, the particle size of the magnetic beads has little effect on the binding performance. The effect of MB-CWBD concentration on the capture performance was also determined, and the results are shown in Figure 3 As shown in b, the binding performance increases with the increase of MB-CWBD concentration. When the concentration of MB-CWBD increases from 0.5 mg / mL to 1 mg / mL, the binding performance slightly increases from 79.08% to 80.78%. -1 The concentration can bind 3.29×10 7 CFU mg -1 BT cells. The results showed that MB-CWBD showed good binding ability to BT strains. Considering the cost, 0.5 mg mL -1 In contrast, MB and MB-BSA only showed very weak binding ability to BMB171 strain ( Figure 3 c), which may be caused by physical adsorption. The binding of MB-CWBD to the BMB171 strain involves the peptidoglycan structure of its cell wall. Specifically, the recognition sites may include three types: the first is the amide bond between the polysaccharide and the peptide in the peptidoglycan; the second is the peptide bond between two amino acids. This recognition site may be particularly present in the stem peptide; and the third is the β-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine. Analysis of the ability of MB-CWBD to bind to Bt showed that MB-CWBD reached saturated binding after 60 minutes of contact with Bt cells. This time was used for subsequent studies, such as Figure 3 As shown in Figure d. After MB-CWBD interacted with the Bt strain for 60 minutes, the exponentially growing Bt strain was resuspended in PBS to form a turbid bacterial suspension. The state of MB-CWBD dispersed in the Bt bacterial solution was a brown suspension. After magnetic adsorption, the Bt bacterial solution became clear. The results showed that MB-CWBD had binding properties to the Bt strain. However, after MB was dispersed in the Bt bacterial solution and adsorbed by a magnet, the bacterial solution remained turbid. The results showed that MB had no binding properties to the Bt strain. Figure 3 The morphology of MB-CWBD was observed by SEM, and the results showed that the Bt strain was successfully bound to the magnetic beads, as shown in Figure e. Figure 3As shown in Figure f. Considering that BT has strong stress resistance and can survive in various adverse environments, the differences in the binding ability of MB-CWBD to Bt at different pH, temperature, and salt concentrations were studied. The results showed that MB-CWBD exhibited high Bt binding ability at pH 4.0, with a binding capacity of 80.55%, and achieved a binding capacity of more than 78% between pH 3.0 and pH 10.0, as shown in Figure f. Figure 3 MB-CWBD achieved a binding capacity higher than 71% at ambient temperature between 20℃ and 35℃, as shown in Figure g. Figure 3 As shown in Figure h, the strongest binding capacity was 78.98% at 28°C. Under conditions with a NaCl concentration lower than 100mM, the binding performance of MB-CWBD to Bt strains was much higher than 73%. When the concentration reached 250mM, the binding performance dropped significantly to 9.56%. Figure 3 As shown in Figure 1. The efficient combination of Bt's pH and temperature tolerance and broad salt tolerance will facilitate the practical application of MB-CWBD in different environments. Therefore, considering cost-effectiveness and ease of operation, it is reasonable to select a concentration of 0.5 mg / mL MB-CWBD for subsequent studies. The MB-CWBD-related parameters used in this study, including magnetic bead size, magnetic bead concentration, concentration of the coupling protein sample, binding time with the indicator strain, and pH range, were all prepared under optimized conditions to obtain MB-CWBD. MB-CWBD has a wide tolerance to pH, temperature, and salinity, can efficiently capture Bt, and is conducive to its practical application in a variety of environments.

[0086] Figure 3 Figure 3 is the result of the binding ability of MB-CWBD to Bt strains, among which, Figure a shows the effect of magnetic beads of different particle sizes on Bt binding ability; Figure b shows the effect of MB-CWBD concentration on binding performance; Figure c shows the binding ability of MB-CWBD, MB-BSA and MB to Bt strains; Figure d shows the dynamic adsorption of MB-CWBD and MB-BSA on Bt strains; Figure e is a schematic diagram of the binding performance of MB and MB-CWBD to Bt strains; Figure f is an SEM image of MB-CWBD binding to Bt; Figure g shows the effect of pH on the ability of MB-CWBD to bind to BT; Figure h shows the effect of temperature on the ability of MB-CWBD to bind to Bt; Figure i shows the effect of NaCl solution concentration on the ability of MB-CWBD to bind to Bt.

[0087] Binding performance of 2.3MB-CWBD against different strains

[0088] The constructed MB-CWBD was used to analyze the binding performance of different strains stored in our laboratory. The results showed that MB-CWBD had a binding capacity of over 83% for the Bacillus strain BMB171, and a binding capacity of 70% to 73% for other Bacillus strains. MB-CWBD also showed high selectivity for strains of the same genus Bacillus, including Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus pumilus, and Bacillus amyloliquefaciens, while the binding capacity for other species outside the genus was less than 16%, such as Figure 4 As shown in Figure a and Table 2. This shows that the magnetic beads constructed in this study have good specificity and strong anti-interference ability.

[0089] Table 2 Test results of binding ability of test strains

[0090] kind serial number Binding performance (%) Bacillus thuringiensis BMB171 83.61±1.54 Bacillus thuringiensis CS 33 70.48±0.58 Bacillus thuringiensis YM03 72.75±0.3 Bacillus cereus ATCC 10987 44.51±0.53 Bacillus cereus ATCC 14579 46.78±0.35 Bacillus subtilis CMCC 63501G3 51.76±0.47 Bacillus anthracis Ba A16R 56.73±0.24 Bacillus pumilus GR8 53.99±0.45 Bacillus amyloliquefaciens GIM 1.403G4 20.91±0.7 Staphylococcus aureus ATCC 6538G3 16.39±0.42 Vibrio mimicus NT 5.84±0.28 Vibrio parahaemolyticus FRX-SJ5 3.53±0.61 Vibrio parahaemolyticus FRX-SJ9 5.06±0.26 Klebsiella odorifera 1519 9.21±0.31 Klebsiella pneumoniae Xu1 7.13±0.63 Klebsiella pneumoniae 1025 10.57±1.16 Escherichia coli CMCC 44102 8.43±0.18 Escherichia coli CMCC 40396 8.95±0.61 Escherichia coli CMCC 40482 7.57±0.23 Acinetobacter baumannii 2AB 4.05±0.28 Acinetobacter baumannii 4AB 3.18±0.23 Salmonella enterica CSV 2.77±0.5 Salmonella enterica RSV 1.35±0.06

[0091] 2.4 Binding performance of CWBD and MB-CWBD on Bacillus thuringiensis spores and vegetative bodies

[0092] Spores are dormant bodies that are formed in the cells of certain bacteria in the late stage of growth and development. They are round or oval, have thick walls, low water content, and strong resistance to stress. The results showed that the concentration of CWBD protein decreased with the extension of the interaction time. The results showed that CWBD protein had a better binding effect on vegetative bodies. At 60 minutes, the concentration of CWBD protein decreased by 32.43% after binding with vegetative bodies, while the protein concentration decreased by 10.81% after binding with spores. BSA protein had no obvious binding effect on vegetative bodies and spores, and the protein concentration only decreased by 5.26%. After CWBD and BSA proteins were in contact with spores and vegetative bodies for 60 minutes, the protein concentration almost stopped decreasing, and the protein binding reached saturation, such as Figure 4 The results show that CWBD protein has a strong binding ability to Bt vegetative bodies. The binding ability of MB-CWBD to Bacillus thuringiensis spores and vegetative bodies was measured. The results showed that the binding rate of MB-CWBD to Bt vegetative bodies reached 80%, while the binding rate to spores was only 20%. Figure 4This is shown in Figure c. This is largely due to the structure of the spore, which consists of the exosporium, spore coat, cortex, and core. The exosporium and spore coat are the first barrier to the spore's defenses against the external environment. The exosporium primarily contains lipoproteins and has poor permeability. The spore coat is composed of various hydrophobic amino acids, accounting for 50%-80% of the total spore protein, protecting the spore from external enzymes, activators, and other substances. The outermost layer of the vegetative body is the cell wall, the primary component of which is peptidoglycan. CWBD and MB-CWBD achieve specific binding through the specific recognition of the lytic enzyme CWBD with the peptidoglycan in the bacterial cell wall. Therefore, CWBD and MB-CWBD exhibit much higher binding abilities to Bt vegetative bodies than to spores.

[0093] 2.5 Isolation of Bacillus thuringiensis strains from simulated soil environments

[0094] In order to analyze the ability of MB-CWBD to bind Bt strains in simulated soil environment, Bacillus subtilis CMCC63501G3, Staphylococcus aureus ATCC 6538G3, and Escherichia coli CMCC 40482 were added to the soil as interference strains. Figure 4 As shown, the binding kinetics of MB-CWBD with Bacillus tumefaciens strain BMB171, Bacillus subtilis CMCC 63501G3, and Escherichia coli CMCC40482 demonstrate significant differences in the binding of MB-CWBD to these strains. At 60 minutes of interaction, MB-CWBD reached saturation binding for each strain, with binding rates of 80.44%, 51.93%, 18.02%, and 8.11% for Bacillus tumefaciens, Bacillus subtilis, Staphylococcus aureus, and Escherichia coli, respectively. These results indicate that complex soil environments have little impact on the binding ability of MB-CWBD to target bacteria and its selectivity, demonstrating the high specificity of the MB-CWBD prepared in this study.

[0095] Therefore, the binding selectivity of MB-CWBD was further analyzed in simulated soil experiments by simultaneously adding Bacillus thuringiensis, Bacillus subtilis, Staphylococcus aureus, and Escherichia coli to sterilized soil. The results showed that over 73.52% of Bacillus therapeutics cells were bound from the soil, a binding rate comparable to that achieved when only one strain was present in the simulated soil. The 43.70% binding rate for Bacillus subtilis was likely due to the similar surface features of Bacillus subtilis cells and Bacillus therapeutics cells. Notably, the recovery efficiencies for Staphylococcus aureus and Escherichia coli were lower, at 15.98% and 7.90%, respectively, further highlighting the specific selectivity of MB-CWBD for Bacillus therapeutics cells.

[0096] Figure 4Figure 3 is the result of the binding selectivity of MB-CWBD to Bacillus thuringiensis strains, where (a) shows the binding ability of MB-CWBD to different strains; (b) shows the dynamic changes in the protein concentrations of CWBD and BSA proteins binding to Bt strain spores and vegetative bodies, respectively; (c) shows the binding performance of MB-CWBD and MB-BSA to Bt spores and vegetative bodies; (d) shows the dynamic adsorption curve of MB-CWBD to Bacillus thuringiensis, Bacillus subtilis, Staphylococcus aureus and Escherichia coli in a simulated soil environment; (e) shows the binding performance of MB-CWBD to Bacillus thuringiensis, Bacillus subtilis, Staphylococcus aureus and Escherichia coli in a simulated soil environment.

[0097] 2.6 Ability to bind Bacillus thuringiensis strains from the natural environment

[0098] The ability of MB-CWBD and traditional isolation methods to bind BT strains from natural soil environments was compared. The results showed that the traditional isolation method could kill 30.17% of the strains in the soil, but the remaining strains accounted for a high proportion of non-target strains, of which Bt strains accounted for only 7%, such as Figure 5 a. In contrast, the MB-CWBD magnetic separation method effectively removed 82.77% of the interfering strains. After MB-CWBD treatment, the remaining bacterial population accounted for 17.23% of the total soil bacteria, of which 36.55% were BT strains ( Figure 5 a). In terms of recovery, the recovery of the MB-CWBD method was 1.29 times that of the heat treatment method, which were 6.29% and 4.89%, respectively. More importantly, the ability of the MB-CWBD magnetic separation method to remove non-target strains was increased by 2.74 times, which can greatly improve the efficiency of obtaining Bt strains. The recovery rate is defined as the ratio of the concentration of Bt strains after treatment to the total concentration of bacteria in the soil. These findings indicate that the MB-CWBD method is significantly superior to the traditional heat treatment method in terms of separation efficiency and selectivity in isolating Bt strains from the natural environment. Observation of the isolated strains by optical and scanning electron microscopy showed that the isolated strains had the characteristics of Bt strains, including rod-shaped morphology, spores and parasporal crystals ( Figure 5 b, c). These observations further support the high specificity of the MB-CWBD magnetic separation method for isolating Bt strains.

[0099] Figure 5 To bind new Bt strains from natural soil; Figure a shows the efficiency of traditional separation methods and MB-CWBD magnetic separation methods in recovering Bt strains from natural soil environments; Figure b is a microscopic observation of bacteria bound by MB-CWBD; Figure c is a SEM image of bacteria bound by MB-CWBD; in the figure, SP represents spores; PC represents paraspore crystals.

[0100] 3. Conclusion

[0101] Efficient screening technologies for functional microorganisms are gaining increasing attention across various fields. However, existing isolation strategies suffer from inefficiency, high cost, and time-consuming processes, making the development of efficient and high-throughput screening strategies highly urgent. This study successfully leveraged the specific binding ability of a bacteriophage cell wall-binding domain to the bacterial cell wall to establish a magnetic bead-based detection and separation method for Bacillus thuringiensis. This method demonstrates high sensitivity, good specificity, strong acid and alkali resistance, and good temperature and salt tolerance, further broadening its application. The MB-CWBD discovered in this study demonstrated efficient, targeted, high-throughput, and time-saving capabilities when binding to Bt strains in simulated natural environments. Even in natural environments, MB-CWBD maintained efficient targeting of Bt strains, demonstrating its broad applicability and potential. The promotion and application of this technology will bring new breakthroughs to the development and utilization of microbial resources, particularly in the screening of Bacillus thuringiensis.

[0102] The above content is a further detailed description of the present application in conjunction with specific implementation methods, and the specific implementation of the present application cannot be considered to be limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A microbial targeting material, characterized in that: The invention comprises a solid phase carrier and a phage cell wall binding domain protein cross-linked on the solid phase carrier.

2. The targeting material according to claim 1, characterized in that: The phage cell wall binding domain protein is the cell wall binding domain protein of the lytic enzyme PlyBt33 of Bacillus thuringiensis phage BtCS33.

3. The targeting material according to claim 2, characterized in that: The cell wall binding domain protein of the lytic enzyme PlyBt33 is the sequence shown in SEQ ID NO.

1.

4. The targeting material according to any one of claims 1 to 3, characterized in that: The solid phase carrier is a magnetic bead, a chip, a porous separation column or a metal nanoparticle; Preferably, the solid phase carrier has a modification group cross-linked with the protein, or a group with a metal ion is introduced into the solid phase carrier, and the metal ion is used to form a specific bond with the recombinant protein containing a histidine tag; Preferably, the metal ions include Ni 2+ 、Co 2+ ; Preferably, the modifying group comprises a hydroxyl group.

5. A recombinant plasmid, characterized in that: Contains a gene fragment that expresses the cell wall binding domain protein of the lytic enzyme PlyBt33 of Bacillus thuringiensis phage BtCS33.

6. The recombinant plasmid according to claim 5, characterized in that: The cell wall binding domain protein of the lytic enzyme PlyBt33 is the sequence shown in SEQ ID NO.1; Preferably, the gene fragment of the cell wall binding domain protein of the lytic enzyme PlyBt33 is the sequence shown in SEQ ID NO.

2.

7. A recombinant cell, characterized in that: Contains the recombinant plasmid according to claim 5 or 6.

8. Use of the targeting material according to any one of claims 1 to 4, the recombinant plasmid according to claim 5 or 6, or the recombinant cell according to claim 7 in screening Bacillus thuringiensis.

9. A kit for screening Bacillus thuringiensis, characterized in that: The method comprises at least one of the targeting material according to any one of claims 1 to 4, the recombinant plasmid according to claim 5 or 6, and the recombinant cell according to claim 7.

10. A method for screening microorganisms, characterized in that: The method comprises contacting the targeting material according to any one of claims 1 to 4 with a sample, and screening the target microorganism from the sample through the binding of the targeting material to the target microorganism; the phage cell wall binding domain protein cross-linked on the solid phase carrier in the targeting material is provided by the phage of the target microorganism.

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