Application of human galectin-3 in prevention and treatment of plutella xylostella

Abstract

The application discloses application of human galectin-3 in prevention and treatment of plutella xylostella, and belongs to the technical field of agricultural pest control. Galectin-3 can specifically bind to the chitin component of the peritrophic membrane of the midgut of plutella xylostella, interfere with the semi-permeable barrier function of the peritrophic membrane, has a significant prevention and treatment effect on plutella xylostella, and the amino acid sequence is shown as NCBI accession number AAA36163. The application further provides a product containing a gene encoding galectin-3, a recombinant expression vector or a recombinant engineering bacterium, and corresponding insecticides and control methods. The insecticide takes human galectin-3 as an effective component, is compounded with an agriculturally acceptable adjuvant, and has a final concentration of 200-400 μg / mL of the effective component. The application is a biological green control product, has specific target points, high safety, no residue, stable control effect, can effectively solve the problems of plutella xylostella resistance and chemical pesticide pollution, and provides a new way for cruciferous vegetable pest green control.

Description

Technical Field

[0001] This invention belongs to the field of agricultural pest and disease control technology, and in particular relates to the application of human galactohemagglutinin-3 in the control of diamondback moth. Background Technology

[0002] Galectins are a family of lectins widely found in animals, plants, and microorganisms. Their core characteristic is the presence of a conserved carbohydrate recognition domain (CRD), which specifically binds to β-galactosidase ligands, thereby participating in various physiological processes such as cell adhesion, immune regulation, and signal transduction. Based on structural differences, the galectin family can be divided into three categories: prototypical, tandem repeat, and chimeric, with the chimeric category containing only Galectin-3.

[0003] In the agricultural field, existing research on Galectin mainly focuses on insect-derived or plant-derived members. Some studies have confirmed that these proteins can participate in the immune recognition process of pests and diseases. However, the application of human Galectin in pest control has not been fully developed, especially in the green control technology system targeting lepidopteran pests, where there is a clear research gap and the potential of related technologies has not been explored.

[0004] Galectin-3, also known as IgE-binding protein, MAC2, or CBP35, is the most structurally unique member of the Galectin family. Composed of 250 amino acid residues, its molecular structure comprises three parts: a short, disordered N-terminal domain (approximately 120 amino acids), a middle collagen-like repeat sequence (containing 9 PGAYPG repeat units), and a conserved C-terminal CRD (approximately 130 amino acids). The C-terminal CRD is the core structure for ligand binding, specifically recognizing the hydroxyl site of galactose through a triangular conformation formed by key amino acids such as arginine and histidine. The N-terminal domain mediates the formation of pentamers and other multivalent complexes, enhancing its cross-linking ability with ligands. This dual-domain collaborative mechanism is crucial for its functional realization. Currently, research on Galectin-3 mainly focuses on mammalian inflammatory responses, fibrotic lesions, and tumor regulation. Its function in agricultural pest and disease control has not been systematically reported, especially whether its unique chimeric structure endows it with binding activity targeting specific insect tissues, which requires further exploration and verification.

[0005] The diamondback moth, belonging to the family Pyrenidae in the order Lepidoptera, is a major global pest of cruciferous vegetables. Its host range covers nearly 300 crop species, including cabbage, kale, and rapeseed. It damages crops by feeding on leaves, tender stems, and young pods, causing yield losses of 30%-80% in severe cases. This pest is characterized by a short development cycle, rapid reproduction rate, and strong environmental adaptability, making it one of the most pesticide-resistant agricultural pests globally. It causes $4-5 billion in direct economic losses to global agricultural production annually, posing a serious threat to the cruciferous vegetable industry.

[0006] Currently, the control of diamondback moth still relies primarily on chemical pesticides. However, the long-term and excessive application of chemical pesticides not only leads to excessive pesticide residues in agricultural products but also causes a series of ecological and environmental problems such as soil and water pollution, which contradicts the concept of green agricultural development. Among existing biological control methods, microbial preparations such as Bacillus thuringiensis (Bt) and Beauveria bassiana have the advantage of being environmentally friendly, but they also have inherent drawbacks such as slow insecticidal action and high dependence on environmental conditions such as field temperature and humidity. Moreover, with long-term application, diamondback moth has evolved a clear resistance mechanism to Bt protein, namely, activating the MAPK signaling pathway through the midgut transcriptional regulatory loop to resist toxicity, further reducing the control efficacy of these preparations. Therefore, developing novel biological control agents with high activity, high stability, and low resistance risk is a key technological requirement to solve the current dilemma of diamondback moth control.

[0007] Given that the C-terminal CRD of Galectin-3 possesses glycoligand-binding activity, and its unique chimeric structure endows it with multivalent binding capability and conformational stability, it holds promise as a novel insect-resistant protein targeting the chitin of the peritrophic membrane of the diamondback moth. Therefore, exploring the application of human Galectin-3 in the control of the diamondback moth and clarifying its mechanism of action will not only fill the research gap in human Galectin in the field of agricultural pest control, but also enrich the green control technology system for the diamondback moth, providing a new technical pathway for the sustainable management of cruciferous vegetable pests, and has significant theoretical and practical value. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of existing technologies and provide the application of human galactolectin-3 in the control of diamondback moth. It aims to break through the ecological risks of chemical control and the efficiency bottleneck of biological control of diamondback moth, provide a new biological source technology solution for the control of diamondback moth, and enrich the agricultural green control system.

[0009] This invention provides the application of human galactolectin-3 in the control of diamondback moth.

[0010] Furthermore, Galectin-3 can specifically bind to the chitinous components of the perifeeding membrane in the midgut of diamondback moth and interfere with the semi-permeable barrier function of the perifeeding membrane.

[0011] Furthermore, the Galectin-3 is the human galactolectin-3 shown in NCBI accession number AAA36163.

[0012] The present invention also provides a product comprising a gene encoding Galectin-3, a recombinant expression vector containing the gene, or a recombinant engineered bacterium containing the recombinant expression vector.

[0013] Furthermore, the product is a biological insecticide or biological control agent.

[0014] The present invention also provides a method for controlling pests, which involves applying the above-mentioned product to pests, their food, or their habitat.

[0015] Furthermore, the pest in question is the diamondback moth.

[0016] The present invention also provides an insecticide for controlling diamondback moth, the active ingredient being the aforementioned human galactohemagglutinin-3, and containing agriculturally acceptable adjuvants.

[0017] Furthermore, the final concentration of the active ingredient in the diamondback moth feed is 200~400 μg / mL.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] The human galactoglobulin-3 used in this invention is a bioactive protein. Compared with traditional chemical pesticides, it is green and environmentally friendly, leaving no residue on crop surfaces and in the growing environment, and will not cause environmental pollution, thus meeting the current development needs of green pest control in agriculture.

[0020] Human galactolectin-3 can specifically bind to the chitin components of the peritrophic membrane of the midgut of diamondback moth. It has a single and highly targeted action site and has no significant adverse effects on non-target organisms and various beneficial insects in the field, thus greatly improving the biosafety of diamondback moth control in the field.

[0021] The control method for diamondback moth is highly targeted. Applying active protein to the surface of diamondback moth food ensures that the moth can effectively feed on it and exert its insecticidal effect. The control effect is clear and stable, effectively making up for the shortcomings of some existing biological control agents that easily lead to resistance in diamondback moths. Attached Figure Description

[0022] Figure 1 Venn diagram showing the results of screening chitin-binding proteins using three computer-aided target prediction methods (SEA, CODD-Pred, and Swiss TargetPrediction);

[0023] Figure 2 SDS-PAGE electrophoresis images of prokaryotic expression and purification products of Galectin family proteins; M: protein molecular weight marker; lane 1: BL21 blank control; lane 2: Galectin-1; lane 3: Galectin-3; lane 4: Galectin-4;

[0024] Figure 3 Immunofluorescence localization map of the midgut of diamondback moth larvae; Figure 3 In the middle, A represents the PBS control treatment; Figure 3 B in the middle represents Galetin-1 processing; Figure 3 C in the middle represents Galetin-3 processing; Figure 3 D in the middle represents Galetin-4 processing;

[0025] Figure 4 Electrophoresis diagram to verify the binding of chitin magnetic beads to Galectin family proteins; M: protein molecular weight marker; Lane 1: bovine serum albumin (BSA) negative control; Lane 2: Galectin-1; Lane 3: Galectin-3; Lane 4: Galectin-4;

[0026] Figure 5 A graph showing the kinetic characteristics of the affinity between Galectin family proteins and chitosan; Figure 5 In Figure A, the binding kinetics curve between Galectin-1 and chitosan is shown. Figure 5 In Figure B, the binding kinetics curve of Galectin-3 and chitosan is shown. Figure 5 In the figure, C represents the binding kinetics curve between Galectin-4 and chitosan;

[0027] Figure 6 Electrophoresis diagram of the stability of Galectin family proteins against midgut enzyme solution in diamondback moth; M: protein molecular weight marker; lane 1: midgut enzyme solution blank control; lane 2: Galectin-1; lane 3: Galectin-3; lane 4: Galectin-4;

[0028] Figure 7 This is a comparison of the insecticidal effects of Galectin family proteins on the third instar larvae of the diamondback moth. 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] Example 1: Screening of chitin-binding proteins using computer-aided reverse docking

[0031] 1. Experimental materials and tools

[0032] (1) Chitin molecular structure file: The basic building block of chitin is N-acetylglucosamine. By logging into the PubChem database (PubChem CID is 24139), the N-acetylglucosamine SMILE file characters can be obtained: CC(=O)NC1C(C(C(OC1O)CO)O)O.

[0033] (2) Reverse docking platform: includes three online tools, namely Swiss Target Prediction, SEA (Similarity Ensemble Approach) and CODD-Pred, whose access addresses are https: / / www.swisstargetprediction.ch / , https: / / sea.bkslab.org / and http: / / coddpred.cadd.zju.edu.cn / respectively.

[0034] (3) Data analysis tools: Venny 2.1 was used to draw Venn diagrams and filter intersection targets, and Excel 2019 was used for the statistics and sorting of target parameters.

[0035] 2. Experimental Methods

[0036] (1) Reverse docking target screening: The SMILE file characters of chitin molecules were submitted to three reverse docking platforms for target prediction. For the Swiss Target Prediction platform, the species parameter was set to general type, the binding mode was selected as default, and the list of predicted target proteins and the corresponding binding confidence were obtained after submitting the ligand molecules. For the SEA platform, the similarity threshold was set to 0.8, and the similarity score and statistical significance P value of the target proteins were obtained after submitting the ligand molecules. For the CODD-Pred platform, the pIC50 prediction threshold was set to 5.0, and the pIC50 prediction value and binding probability of the target proteins were obtained after submitting the ligand molecules.

[0037] (2) Target result integration and cross-validation: The target protein lists output by the three platforms were collected, and Venn diagrams were drawn using the Venny2.1 tool to screen the intersection targets predicted by the three platforms. At the same time, the key parameters of the intersection targets were extracted, including binding confidence, P value and pIC50 predicted value. High-confidence targets were screened and sorted according to the criteria of binding confidence > 0.8, P value < 0.01 and pIC50 predicted value > 6.0.

[0038] 3. Experimental Results

[0039] The prediction results from the three platforms differed. The Swiss Target Prediction platform predicted 9 potential targets, the SEA platform predicted 10, and the CODD-Pred platform predicted 5. A Venn diagram clearly shows the overlap of predicted targets across the three platforms, identifying one overlapping target – the Galectin family proteins, including Galectin-1, Galectin-3, and Galectin-4. Statistical analysis of the parameters of the overlapping target revealed that Galectin-3 had the best parameters across all categories. Its binding confidence in Swiss Target Prediction was 0.92, the highest among all overlapping targets; its p-value in SEA was 2.3 × 10⁻⁶. -6 The target met the screening criterion of p-value < 0.01; its pIC50 prediction value in CODD-Pred was 6.8, exceeding the threshold requirement of 6.0. The entire process of target screening, cross-validation, and high-confidence target identification is detailed below. Figure 1 As shown.

[0040] Example 2: Prokaryotic expression and purification of Galectin family proteins

[0041] 1. Expression vector construction and clone identification

[0042] Based on computer-aided target prediction and sequence alignment analysis, Galectin-1, Galectin-3, and Galectin-4 were selected as candidate molecules in this embodiment. The mRNA sequences corresponding to these three galactohemagglutinins could be obtained from the NCBI database. Specifically, the accession number for Galectin-1 is AAA36170, for Galectin-3 it is AAA36163, and for Galectin-4 it is BAA22165. In this embodiment, Nanjing Qingke Biotechnology Co., Ltd. was commissioned to complete the synthesis and molecular cloning of the above genes.

[0043] The coding genes of Galectin protein family members (Gal-1 / 3 / 4) were directionally cloned into the pET-26b(+) prokaryotic expression vector using a double enzyme digestion system (NcoI / XhoI) and a homologous recombination strategy. After colony PCR screening and sequencing confirmation, the recombinant plasmid pET-26b(+)-Gal-1 / 3 / 4 carrying the target gene was finally obtained. Its genetic characteristics include the T7 promoter regulatory element and kanamycin resistance selection markers.

[0044] 2. Prokaryotic expression and protein purification

[0045] (1) Bacterial culture and induced expression

[0046] A single clone of *E. coli* strain BL21(DE3) containing the recombinant expression vector pET-26b(+)-Gal-1 / 3 / 4 was selected and inoculated into 10 mL of 2×YT liquid medium supplemented with 50 μg / mL kanamycin. The medium was then placed in a constant temperature shaking incubator (37℃, 250 rpm) for primary amplification. The turbidity of the bacterial culture was monitored periodically using a UV spectrophotometer. When the optical density (OD600) reached the exponential growth phase of 0.6-0.8, IPTG inducer (isopropyl galactothioglycoside) was added to the culture medium to a working concentration of 1 mM. The culture was induced for 16 hours under the same temperature and shaking conditions to promote efficient expression of the recombinant protein. Immediately after induction, the bacterial culture was transferred to pre-cooled centrifuge tubes and centrifuged at 6000×g for 10 min at 4℃ to collect the bacterial pellet.

[0047] (2) Protein denaturing electrophoresis (SDS-PAGE) detection

[0048] The recombinant bacterial suspension (20 μL) induced by IPTG was vortexed with 4×SDS-PAGE loading buffer (7 μL) and incubated in a boiling water bath for 10 min to allow complete protein development and denaturation. A pre-cast gel (Tris-MOPS buffer system) with a 15% separating gel concentration was used for vertical electrophoresis in a Mini-PROTEAN Tetra electrophoresis tank. The loading volume was 15 μL / well, and electrophoresis was performed at a constant voltage of 140 V for 60 min. The electrophoresis buffer consisted of 25 mM Tris, 192 mM glycine, and 0.1% SDS (pH 8.3). After electrophoresis, the gel was immersed in Coomassie Brilliant Blue R-250 staining solution and stained on a shaker at room temperature for 30 min to develop protein bands. The gel was then repeatedly rinsed with destaining working solution (a mixture of 40% methanol and 10% acetic acid) until the gel background was transparent and the bands were clear. The location of the target protein bands was confirmed by comparison with molecular weight standards.

[0049] (3) Western blot analysis of proteins

[0050] Protein samples separated by SDS-PAGE were transferred to PVDF membranes using a wet transfer system. The transfer conditions were set at 60 V for 90 min. The transfer buffer formulation contained 25 mM Tris base, 192 mM glycine, and 20% methanol (pH 8.3). After transfer, the PVDF membrane was immersed in TBS-T blocking buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.6) containing 5% skim milk powder and blocked on a horizontal shaker at room temperature for 2 h to block non-specific binding sites. Subsequently, the membrane was incubated with anti-His-tagged mouse monoclonal secondary antibody (1:5000 diluted in blocking buffer) at room temperature for 1 h. Unbound antibodies were removed by washing three times (5 min / wash) on a shaker with TBS-T buffer (0.1% Tween-20). Finally, ECL chemiluminescent substrate (premixed solution A and solution B at a 1:1 volume ratio) was uniformly added, and specific signals were captured using a chemiluminescence imaging system in a dark room.

[0051] (4) Nickel column affinity purification of recombinant protein

[0052] The purification of His-tagged proteins was performed using the ÄKTA pure fully automated chromatography system. The specific operation procedure is as follows:

[0053] ① Column equilibration pretreatment: Connect the HiTrap™ Ni-NTA affinity chromatography column (5 mL column bed volume) to the chromatography system and equilibrate the column with binding buffer (20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4). Set the flow rate to 5 mL / min and continue washing until the UV 280 nm absorbance value stabilizes.

[0054] ② Sample loading and adsorption: The supernatant of the bacterial cell lysate after ultrasonic disruption is sterilized through a 0.22μm filter membrane and loaded three times at a flow rate of 1 mL / min. The target capture is achieved by utilizing the specific chelation between the His tag protein and the solid nickel ions.

[0055] ③ Non-specific elution: Switch to washing buffer (20 mM sodium phosphate, 500 mM NaCl, 50 mM imidazole, pH 7.4) and wash the column with 10 column volumes to effectively remove weakly bound contaminating proteins.

[0056] ④ Gradient elution collection: The target protein was dissociated using linear gradient elution buffer (20-500 mM imidazole, 10 CV), and the UV absorption curve was monitored in real time. The eluted components were collected in separate tubes according to the peak characteristics.

[0057] Utilizing the metal chelating property of the histidine tag (His-tag) with nickel ions, the target protein was purified using a Ni-NTA agarose chromatography column (5 mL column volume). Figure 2 As shown, the molecular weights of Gal-1, 3, and 4 were 20.8 kDa, 31.2 kDa, and 38.5 kDa, respectively, as verified by SDS-PAGE and Western blot (1:5000 dilution of anti-His tag monoclonal antibody), which are consistent with the theoretical values.

[0058] Example 3: Immunofluorescence staining combined with localization analysis of Galectin family proteins

[0059] 1. Biological sample pretreatment protocol

[0060] (1) Sample stabilization treatment: After collecting midgut tissue from healthy diamondback moth larvae, the tissue was immediately immersed in 4% formaldehyde polymer fixative (phosphate buffer system pH 7.4) pre-cooled to 4°C and continuously shaken in a constant temperature shaker (4°C, 80 rpm) for 36 hours to ensure the integrity of the tissue morphology.

[0061] (2) Gradient dehydration procedure: After fixation, the sample is treated with an increasing concentration of ethanol (80%, 95%, anhydrous), with each gradient treatment lasting 3 minutes to achieve complete dehydration. Then, it is transferred to xylene clearing agent for two infiltrations (5 minutes each time). The entire process is strictly timed (total time ≤ 12 minutes) to prevent tissue brittleness from affecting the quality of subsequent sections.

[0062] 2. Improvements in microscopy preparation techniques

[0063] (1) Embedding medium infiltration: The dehydrated sample was subjected to three embedding medium (Paraplast X-tra) infiltration treatments (60 minutes each) in a constant temperature wax impregnation instrument (65℃), and then transferred to a pre-cooling mold (-20℃ cooling table) to form standardized wax blocks.

[0064] (2) Precision sectioning process: 5μm continuous sections were prepared using a semi-automatic sectioning system. After being spread in an ultrapure water bath at 42℃, the sections were transferred to polylysine-coated glass slides and cured at 65℃ for 45 minutes to enhance tissue adhesion.

[0065] 3. Antigen epitope activation strategy

[0066] (1) Thermally induced epitope repair: Place the slide in the antigen repair solution (pH 6.0 citrate buffer) and treat it with a high-pressure thermal repair instrument (121℃, 103.4 kPa) for 90 seconds. Immediately transfer it to a buffer solution pre-cooled to 4℃ to implement gradient cooling (cooling gradient 5℃ / min) to maintain the spatial conformation of the antigen epitope.

[0067] (2) Non-specific site blocking: After rinsing three times with PBS buffer (0.01 M, pH 7.4) (5 minutes each time), the site was incubated at room temperature for 90 minutes with blocking solution containing 5% skim milk, which effectively reduced background signal interference.

[0068] 4. Molecular probe localization system

[0069] (1) Primary antibody specific binding: His tag specific primary antibody (HRP-coupled type, 1:800 diluted in blocking solution) was used to carry out the targeted binding reaction for 75 minutes in a humidity-controlled incubator (37℃). The antibody solution was pretreated with a 0.22μm microporous membrane to eliminate complex interference.

[0070] (2) Fluorescence signal amplification: After washing with PBST (containing 0.1% Triton X-100) three times (8 minutes each time), FITC-labeled secondary antibody (1:1500 dilution) was introduced and reacted in a dark environment (25℃) for 50 minutes. A blank control was set up simultaneously to exclude non-specific staining.

[0071] 5. Microscopic imaging preprocessing

[0072] Elimination of endogenous interference: Adding a compound autofluorescence inhibitor (containing 0.3% Sudan Black B) and treating for 45 minutes in the dark significantly reduced endogenous pigment interference in the midgut tissue. Figure 3 The biodistribution study based on fluorescence confocal microscopy shows that members of the galectin family (Gal-1 / 3 / 4) exhibit differential targeting and binding characteristics in the digestive system of lepidopteran insects. The experiment used a standard feeding method, delivering feeds containing different galectin proteins to third-instar larvae of the diamondback moth, and histological analysis was performed using a Leica microscopy system.

[0073] Cross-sections of the midgut in the control group (PBS-treated) showed intact epithelial layer arrangement and clear basement membrane structure; no non-specific signals were detected in the fluorescence channel. Figure 3 A). In stark contrast, the Galectin-1 treatment group ( Figure 3 B) and Galectin-3 treatment group ( Figure 3 High-intensity fluorescent aggregation was observed between columnar epithelial cells in group C. Galectin-4 treatment group ( Figure 3 D) Although the fluorescence signal intensity was significantly reduced, its pathological features were more pronounced: the midgut microvilli structure disintegrated and the peritrophic membrane showed irregular breaks, suggesting that the protein may affect the insect digestive system through a mechanism of action different from Galectin-1 / 3.

[0074] Example 4: Verification of the combination of chitin magnetic beads and protein coprecipitation

[0075] 1. Magnetic bead pretreatment and reaction system construction

[0076] Accurately measure the predetermined volume of chitin magnetic bead suspension (Chitin Magnetic Beads, part number: S6651) according to the reaction conditions, and strictly follow the manufacturer's technical manual to perform magnetic bead pretreatment. The operation procedure is as follows:

[0077] (1) Washing of magnetic microspheres: Take an appropriate amount of chitin magnetic bead suspension and transfer it into a 1.5 mL centrifuge tube and place it on a magnetic rack for 60 s. After the solid and liquid phases are completely separated, remove the liquid phase.

[0078] (2) Buffer system equilibration: Add 1 mL of pre-cooled PBS (pH 7.4) to the treated magnetic microspheres, gently vortex for 10 s, and repeat the magnetic separation operation. This washing process is repeated three times to ensure that the stable components in the storage buffer are completely removed.

[0079] 2. Protein-magnetic bead binding reaction

[0080] (1) Preparation of reaction system: Take 300 μL of 200 μg / mL galactose lectin protein (Galectin-1 / 3 / 4) and bovine serum albumin (BSA, negative control) solution respectively, and mix them with an equal volume of pretreated magnetic bead suspension in a 1.5 mL centrifuge tube.

[0081] (2) Reaction conditions: The mixture was transferred to a 360° omnidirectional rotary mixer (speed: 20 rpm) and incubated at 4°C for 24 h to ensure the full binding of the protein to the ligands on the surface of the magnetic beads.

[0082] 3. Magnetic bead separation and washing

[0083] (1) Magnetic bead separation: Transfer the reaction mixture into a 1.5 mL centrifuge tube, fix it vertically on a magnetic rack, and allow it to stand at room temperature for 120 s to allow the magnetic microspheres to fully aggregate on the tube wall. After a clear boundary is formed at the magnetic bead-liquid interface, use a 200 μL micropipette that has been certified by metrology to slowly aspirate the liquid phase component along the tube wall at a 15° angle. During the operation, strictly maintain a distance of ≥2 mm between the tip of the pipette and the magnetic bead layer to prevent physical interference.

[0084] (2) Washing procedure: Add 1 mL of PBS buffer (PBST) containing 0.1% Tween-20 to each tube, vortex for 2 min to fully suspend the magnetic beads, then magnetically separate and discard the washing solution. Repeat this step 5 times to standardize the washing parameters to inhibit nonspecific binding.

[0085] (3) Verification of washing effect: After the last wash, the supernatant was retained and the washing solution was tested by SDS-PAGE (15% separating gel, 140 V constant voltage electrophoresis for 60 min) to check whether the target protein was residual in the washing solution, and to confirm the washing effectiveness.

[0086] Based on the principle of solid-phase adsorption, chitin-coated magnetic beads were used to assess the specific binding ability of Galectin protein. Galectin-1 / 3 / 4 and BSA (negative control) were co-incubated with magnetic bead suspensions (25℃, 1 h) using 20 mM phosphate buffer (pH 7.4, containing 150 mM NaCl). After removing non-binding proteins using magnetic separation, the binding ability was analyzed by SDS-PAGE.

[0087] Experimental results are as follows Figure 4 As shown, the magnetic bead adsorption rates of the Galectin-1 / 3 / 4 treatment groups were 15.6±2.1%, 32.3±3.7%, and 10.8±1.9%, respectively, while the BSA control group only had a rate of 3.2±0.8%. One-way ANOVA confirmed that the binding efficiency of Galectin-3 was significantly higher than that of other subtypes, and the difference between the experimental group and the control group was statistically significant (P<0.05), indicating that members of the Galectin family have specific chitin-binding capabilities, with Galectin-3 exhibiting the best affinity.

[0088] Example 5: Determination of Molecular Affinity using Biolayer Interferometry (BLI)

[0089] This embodiment utilizes a real-time bio-layer interferometry (BLI; Sartorius Octet® RED 96e, Germany) system to study molecular binding dynamics using a dip and read detection mode. During the experiment, biotin-labeled target proteins were immobilized on the surface of an amino-reactive sensor (AR2G Biosensor). A 0.05% Tween-20 PBS buffer solution was used as the operating system, with the temperature controlled at 25℃ ± 0.5℃ and the oscillation frequency maintained at 1000 rpm. Ligand gradient concentration solutions (0.78-100 nM) were sequentially loaded using an automated sample introduction system, and the optical thickness changes of the binding phase (300 s) and dissociation phase (600 s) were recorded in real time.

[0090] The raw sensor data was fitted with a third-order polynomial and corrected for the reference channel using Octet® Analysis Suite (version 11.1.0.33). The molecular association rate constant (kon, unit: M-1s-1) and dissociation rate constant (koff, unit: s-1) were calculated using a 1:1 Langmuir combined model. The equilibrium dissociation constant (unit: nM) was then derived based on the formula KD=koff / kon.

[0091] 1. Construction of metal chelate sensing interface

[0092] A commercially available HIS1K biosensor (ForteBio, Germany) was used, and a standardized pretreatment procedure was performed: the sensor array was vertically immersed in 10 mM PBS buffer (pH 7.4, containing 150 mM NaCl) and continuously activated for 15 ± 0.5 min in a constant-temperature mixer (25℃, 200 rpm). This process achieves Ni activation through coordination chemistry principles. 2+ The directional deposition of ions on the NTA-modified chip surface forms a high density of metal affinity sites (approximately 3.2 × 10^11 sites / cm² per sensing point). 2 After activation, the sensor is rinsed three times with ultrapure water, dried with nitrogen, and then stored in a desiccator for later use.

[0093] 2. Development of anti-interference liquid phase systems

[0094] To reduce nonspecific binding effects, a dual stabilizer was introduced into the basal PBS buffer (10 mM sodium phosphate, 137 mM NaCl, 2.7 mM KCl): 0.1% (w / v) bovine serum albumin (BSA, Sigma-Aldrich A7906) was used to shield against hydrophobic interactions. The buffer was filtered through a 0.22 μm PVDF membrane and stored at 4°C protected from light for no more than 72 h. Galectin isoforms (Gal-1 / 3 / 4) were diluted to a final concentration of 20 μg / mL using this buffer system, centrifuged sterilly (12000 g, 10 min, 4°C), and used immediately.

[0095] 3. Four-step dynamic detection scheme

[0096] To reduce nonspecific binding effects, a dual stabilizer was introduced into the basal PBS buffer (10 mM sodium phosphate, 137 mM NaCl, 2.7 mM KCl): 0.1% (w / v) bovine serum albumin (BSA, Sigma-Aldrich A7906) was used to shield against hydrophobic interactions. The buffer was filtered through a 0.22 μm PVDF membrane and stored at 4°C protected from light for no more than 72 h. Galectin isoforms (Gal-1 / 3 / 4) were diluted to a final concentration of 20 μg / mL using this buffer system, centrifuged sterilly (12000 g, 10 min, 4°C), and used immediately.

[0097] The experimental plate (96-well black microplate, Greiner Bio-One) was designed with the following sections: Baseline Equilibration: The probe was immersed in the running buffer for 180 s, and signal stability was monitored (allowable fluctuation range ±0.1 nm), ensuring baseline drift <0.005 nm / s. Target Protein Loading: His-tagged proteins (Gal-1 / 3 / 4, concentration 20 μg / mL) were added to the second row of wells, and the probe was immersed in the solution for ligand capture (Loading Phase) for 300 s (signal saturation threshold Δλ = 1.5 nm). After loading, the probe was transferred to the running buffer for another 180 s to equilibrate and remove weakly binding proteins.

[0098] Binding kinetics (Association): A gradient dilution of chitin / chitosan solution was placed in the fourth row of wells. The probe was sequentially immersed in each well to initiate the association phase, which lasted 600 s. Changes in biolayer thickness (Δλ, nm) were recorded in real time at a sampling frequency of 10 Hz. Dissociation kinetics (Dissociation): After binding, the probe was returned to the running buffer for dissociation monitoring (Dissociation Phase), which lasted 600 s, to assess the stability of the complex.

[0099] The binding kinetics of Galectin-1, Galectin-3, and Galectin-4 proteins to natural chitin and its deacetylated product chitosan were systematically determined using the Octet Red96 detection platform based on the principle of biomembrane interference. The experimental results are as follows: Figure 5 As shown, all three classes of Galectin proteins exhibited specific binding activity to two chitin derivatives. Galectin-1 and Galectin-3 showed significantly higher affinity for both ligands than Galectin-4, with binding strength differences of 4.8–5.2 times. Notably, despite differences in binding efficiency among the different isoforms, all tested proteins exhibited molecular characteristics specific to the recognition of β-1,4-glycosidic bonds, suggesting that the Galectin family may interact with peritrophic membrane chitin through conserved domains.

[0100] Example 6 Study on the enzymatic resistance of Galectin family proteins

[0101] 1. Optimization of insect digestive fluid preparation process

[0102] (1) Sample pretreatment process: Fourth-instar diamondback moth larvae of the same developmental stage were selected and subjected to 24-hour fasting treatment to empty the contents of the digestive tract. Under low temperature conditions, the larvae were washed three times by centrifugation (800×g, 60 s each time) with a sodium chloride solution containing 1 mM benzyl sulfonyl fluoride pre-cooled to 4℃ to effectively remove contaminants from the body surface.

[0103] (2) Digestive system separation technique: Midgut tissue samples were rapidly dissected using microsurgical instruments on a low-temperature operating platform. The collected biological materials were immediately transferred to a pre-cooled preservation tube (1.5 mL, Eppendorf brand), and 1 mL of tissue lysis buffer (0.15 mol / L NaCl, 50 mmol / L Tris-HCl pH 7.4, 1 mmol / L ethylenediaminetetraacetic acid) was added for low-temperature preservation.

[0104] (3) Cell disruption and component separation: Tissue disruption was performed using an ultrasonic lysing device (30% output power, pulse mode: 1 second working / 2 seconds intermittent, 3 cycles). The clear supernatant was obtained by high-speed low-temperature centrifugation (4℃, 12000×g, 15 min).

[0105] (4) Enzyme activity preservation method: Add 10% glycerol (volume fraction) as a cryoprotectant, aliquot and store in an ultra-low temperature freezer (-80℃) for long-term storage. Before use, perform rapid rewarming treatment (37℃ water bath for 30 seconds), and enzyme activity detection confirms that the activity retention rate exceeds 90%.

[0106] 2. Establishment of an in vitro biomimetic digestion model

[0107] (1) Construction of reaction environment: A 50 mM / L sodium carbonate buffer system (pH 10.5) was selected to simulate the alkaline microenvironment of the hindgut of the target insect. The buffer was sterilized by a 0.22 μm filter membrane and then pre-cooled to the experimental temperature.

[0108] (2) Protein pretreatment: Galectin-1 / 3 / 4 recombinant protein (concentration 1 mg / mL) was replaced with a molecularly cut-off membrane (10 kDa, Spectra / Por brand) to eliminate the interference of ionic strength on the enzymatic hydrolysis reaction.

[0109] (3) Bionic reaction system: A 100 μL standard reaction system was constructed, containing 100 μg of target protein, gradient enzyme solution (0.2-2 μL) and buffer replenishment. Triple biological replicates were set up for each experimental group.

[0110] (4) Dynamic digestion process: The digestion was simulated by continuous incubation in a constant temperature shaking incubator (25℃, 200 rpm) for 12 hours. At the end of the reaction, a protease inhibitor (1 mM PMSF) and a metal chelating agent (10 mM EDTA) were added simultaneously. After standing at low temperature for 10 min, the mixture was quickly frozen (-80℃) for subsequent detection.

[0111] This embodiment uses classic protein electrophoresis to systematically evaluate the enzymatic stability of galectin family members. Under standard electrophoresis conditions (constant voltage 120V, electrophoresis time 60min), a serial dilution experiment revealed that when the sample was diluted to 1:100, all three proteins, Galectin-1, Galectin-3, and Galectin-4, exhibited significant band dispersion, indicating a protease-mediated degradation process.

[0112] In detection at higher dilution gradients (1:1000), the gel imaging system provides quantitative analysis such as... Figure 6 As shown, Galectin-3 protein maintained 72.3 ± 5.1% of its band integrity (n=3), while the band signal intensities of Galectin-1 and Galectin-4 decreased to below the detection threshold (<8.5%, p<0.01). This differential degradation kinetics suggests that Galectin-3 may achieve stronger conformational stability through its unique domains (such as the dimerization tendency of the sugar recognition domain), thereby maintaining structural integrity in proteolytic environments.

[0113] Example 7: Indoor bioactivity determination of diamondback moth

[0114] The three galactoglobulin proteins Galectin-1, 3, and 4 were dissolved in phosphate-buffered saline (PBS, pH 7.4) and the final protein concentration was quantitatively adjusted to 0.3 mg / mL.

[0115] (1) Feed coating: Take 1 mL of protein solution of various concentrations and evenly cover the surface of artificial feed in polystyrene petri dish (Φ=90 mm). Let it air dry naturally in a sterile operating table at 25°C for 12 hours to form a uniform protein coating layer.

[0116] (2) Larval grouping: 30 healthy diamondback moth larvae with synchronous development were selected and randomly divided into: experimental group: Galectin-1 / 3 / 4 treatment group; control group: PBS buffer treatment group.

[0117] The bioactivity of galactolectin was evaluated using the standard dose method, with the working concentration of recombinant protein set at 300 μg / mL. Toxic feed was prepared using a surface coating method (1 mL of treatment solution was applied to each petri dish, total dose 300 μg), and fed to third-instar diamondback moth larvae (30 larvae per group). Mortality was calculated using the corrected formula: (Number of deaths in the treatment group – Number of natural deaths in the control group) / (Total number of larvae – Number of deaths in the control group) × 100%.

[0118] like Figure 7 As shown, the corrected mortality rate in the Galectin-3 treatment group reached 31.6% on day 3 (original mortality rate 35%) and increased to 57.9% on day 5 (original mortality rate 60%), significantly higher than other treatment groups (P<0.001). The Galectin-1 group showed a dose-response relationship, with the corrected mortality rate increasing from 21.1% on day 3 to 36.1% on day 5; the corrected mortality rates in the Galectin-4 groups were 17.5% and 28.3%, respectively. One-way ANOVA showed highly significant differences between each treatment group and the blank control (corrected mortality rate 0%) (P<0.005), confirming that Galectin-3 has the best insecticidal efficacy.

[0119] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. Application of human galactoglobulin-3 in the control of diamondback moth.

2. The application according to claim 1, characterized in that, Galectin-3 can specifically bind to the chitinous components of the perifeeding membrane in the midgut of diamondback moth and interfere with the semi-permeable barrier function of the perifeeding membrane.

3. The application according to claim 1, characterized in that, The Galectin-3 mentioned is the human galactolectin-3 indicated by NCBI accession number AAA36163.

4. A product characterized in that, The product comprises the gene encoding Galectin-3 as described in claim 1, a recombinant expression vector containing the gene, or a recombinant engineered bacterium containing the recombinant expression vector.

5. The product according to claim 4, characterized in that, The product is a biological insecticide or biological control agent.

6. A method for controlling pests, characterized in that, Apply the product of claim 4 or 5 to pests, their food, or their habitat.

7. The method for controlling pests according to claim 6, characterized in that, The pest in question is the diamondback moth.

8. An insecticide for controlling the diamondback moth, characterized in that, The active ingredient is the human galactoglobulin Galectin-3 as described in claim 1, and it contains agriculturally acceptable adjuvants.

9. The insecticide according to claim 8, characterized in that, The final concentration of the active ingredient in the diamondback moth feed is 200-400 μg / mL.

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