A method for preparing chlorantraniliprole nanobodies

Abstract

This invention discloses a method for preparing chlorantraniliprole nanobodies, belonging to the field of biorecognition material preparation for pesticide residue detection. The method involves screening chlorantraniliprole-specific nanobodies using bioinformatics analysis, cloning them into the pET-22b(+) expression vector, transforming them into *E. coli* BL21(DE3) competent cells, and achieving soluble expression of the nanobodies via IPTG induction. High-purity chlorantraniliprole nanobodies are then obtained through periplasmic purification combined with nickel column affinity chromatography. This invention eliminates the need for animal immunization experiments such as alpaca screening through bioinformatics, significantly shortening the nanobodies preparation cycle and reducing costs. The use of the pET-22b(+) prokaryotic expression system combined with an 18℃ low-temperature induction strategy achieves soluble expression of chlorantraniliprole nanobodies. After induction, the target protein band can be clearly detected by SDS-PAGE electrophoresis (see Figure 1), improving the correct folding ratio of the antibody.

Description

Technical Field

[0001] This invention relates to the field of biorecognition material preparation technology for pesticide residue detection, and in particular to a method for preparing chlorantraniliprole nanobodies. Background Technology

[0002] Chlorantraniliprole is a highly effective and low-toxicity o-aminobenzoamide insecticide that kills pests by activating ryanodine receptors in insects and is widely used in agricultural pest control. With its increasing use, chlorantraniliprole residues in agricultural products and the environment have raised concerns about food safety and ecological risks, making the development of rapid and sensitive residue detection methods crucial.

[0003] Currently, pesticide residue detection mainly relies on chromatography and coupled techniques. While these methods offer high accuracy, they require expensive instruments and complex pretreatment processes, making them unsuitable for rapid on-site screening. Immunoassays, with their advantages of ease of operation and low cost, have become an important supplement, with high-performance antibodies being the core of immunoassays. Traditional polyclonal and monoclonal antibodies have long preparation cycles, poor stability, and rely on animal immunization. Although conventional nanobody preparation offers advantages such as small molecular weight and high stability, it still requires alpaca immunization, making the process cumbersome and time-consuming.

[0004] Therefore, there is a need for a method to prepare chlorantraniliprole nanobodies efficiently without animal immunization, which is of great significance for promoting the development of rapid detection technology for its residues. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing chlorantraniliprole nanobodies, which solves the problems mentioned in the background section.

[0006] Technical Solution: To solve the above-mentioned technical problems, according to one aspect of the present invention, more specifically, a method for preparing chlorantraniliprole nanobodies includes the following steps: S1, Nanobody Sequence Screening Specific nanobody sequences for chlorantraniliprole (CYR) were obtained through bioinformatics analysis and screening.

[0007] S2, Expression Vector Construction The selected nanobody genes were cloned into the pET-22b(+) prokaryotic expression vector, and the pET22b-CYR nanobody inducible expression vector was constructed by the biotechnology company.

[0008] S3, bacterial culture and induced expression The pET22b-CYR expression vector was transformed into Escherichia coli BL21(DE3) competent cells to obtain the recombinant strain pET22b-CYR E. coli BL21(DE3); The recombinant strain was streaked onto LB-Amp plates and incubated overnight at 37°C. Select positive single colonies and inoculate them into 50 mL of LB liquid medium containing ampicillin, and incubate overnight at 37°C in a shaker. Take 1 mL of the above culture medium and inoculate it into 125 mL of LB medium containing ampicillin. Incubate at 37°C and 200 r / min with shaking until the OD600 value reaches about 0.6. Add IPTG to the culture medium to a final concentration of 0.1 mmol / L and induce at 18°C ​​for 18-20 h; The induced cell pellet was collected by centrifugation at 4℃ and 5000g for 10 min. After induction, the target protein band was detected at approximately 12 kDa by SDS-PAGE electrophoresis (see [link to SDS-PAGE]). Figure 1 This is consistent with the expected molecular weight of the nanobody.

[0009] S4, Nanobody Purification Periplasmic purification: The cell pellet was resuspended in 3.75 mL of pre-cooled TES buffer and incubated on an ice-cold rocker for 1.5 h; 7.5 mL of TES / 4 buffer was added and incubated on an ice-cold rocker for another 45 min; centrifuged at 12000 g at 4 °C for 30 min and the supernatant was collected as the periplasmic extract. Nickel column affinity chromatography purification: The recombinant nanobodies with His tags in the periplasmic extract were purified by nickel column affinity chromatography to obtain high-purity chlorantraniliprole nanobodies.

[0010] The beneficial effects of the chlorantraniliprole nanobody preparation method of the present invention are as follows: (1) The present invention screens antibody sequences through bioinformatics, eliminating the need for animal immunization experiments such as alpacas, which greatly shortens the preparation cycle of nanobodies and reduces the preparation cost; This invention utilizes the pET-22b(+) prokaryotic expression system combined with a low-temperature induction strategy at 18℃ to achieve soluble expression of chlorantraniliprole nanobodies. After induction, the target protein band can be clearly detected by SDS-PAGE electrophoresis (see [link]). Figure 1 This improved the correct folding ratio of the antibody; (2) The half-maximal inhibitory concentration (IC50) of the nanobody prepared in this invention against chlorantraniliprole is 1.04 μg / mL (see [reference]). Figure 2 It has high sensitivity; after treatment at 90℃ for 30 minutes, it still retains more than 80% of its activity (see...). Figure 4 It also exhibits good tolerance to organic solvents such as methanol and acetone (see [link]). Figure 3 The activity is highest at pH 7.4 (see [link]). Figure 5 The binding capacity is optimal at an ion concentration of 10 mmol / L (see [link to relevant documentation]). Figure 6 Excellent environmental adaptability; (3) Homology modeling analysis showed that 95.2% of the atoms of the nanobody prepared in this invention were distributed in the reasonable region of the Ramachandran diagram (see [reference]). Figure 7 Molecular docking shows that it can form hydrogen bonds and hydrophobic interactions with CYR small molecules (see [link]). Figure 8 , Figure 9 It has strong specificity and can be used as a core recognition material for the development of immunochromatographic test strips or biosensors for chlorantraniliprole residues. Attached Figure Description

[0011] The present invention will now be described in further detail with reference to the accompanying drawings and specific implementation methods.

[0012] Figure 1 SDS-PAGE electrophoresis images of pET22b-CYR E. coli BL21(DE3) after induction, where M is the marker, 1 is E. coli BL21(DE3), 2 is uninduced pET22b-CYR E. coli BL21(DE3), and 3 is induced pET22b-CYR E. coli BL21(DE3). Figure 2 The graph shows the sensitivity assay results for CYR nanobodies. Figure 3 The diagram shows the tolerance of CYR nanobodies to different organic solvents, where A is methanol, B is N,N-dimethylformamide, C is dimethyl sulfoxide, D is acetone, and E is acetonitrile. Figure 4 The graph shows the thermal stability of CYR nanobodies. Figure 5 The inhibition curves of CYR nanobodies at different pH values ​​are shown. Figure 6 The inhibition curves of CYR nanobodies under different ionic intensities are shown. Figure 7 This is the Ramachandran Plot (a quality assessment plot for nanobody homology modeling). Figure 8 A 3D visualization of the docking between anti-CYR nanobodies and CYR small molecules; Figure 9 Visualization (2D) of the docking between anti-CYR nanobodies and CYR small molecules. Detailed Implementation

[0013] The present invention will be described in detail below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the present application can be combined with each other.

[0014] To make the technical solution of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Example

[0015] Reference Figures 1-9 A method for preparing chlorantraniliprole nanobodies, the specific steps of which are as follows: Nanobody sequence screening Specific nanobody sequences for chlorantraniliprole (CYR) were obtained by screening using bioinformatics methods.

[0016] Construction of expression carrier The above-mentioned nanobody gene was cloned into the pET-22b(+) expression vector, and the pET22b-CYR nanobody inducible expression vector was constructed by Nanjing Genscript Biotech Co., Ltd.

[0017] Bacterial culture and induced expression The pET22b-CYR E.coli BL21(DE3) strain was streaked onto LB-Amp plates and incubated overnight at 37°C. Pick a single colony and inoculate it into 50 mL of LB liquid medium containing ampicillin, and incubate overnight at 37°C and 200 rpm on a shaker. Take 1 mL of culture medium and inoculate it into a 250 mL Erlenmeyer flask containing 125 mL of ampicillin LB medium. Incubate at 37 °C and 200 r / min for 2.5 h with shaking until the OD600 value is 0.6. IPTG was added to a final concentration of 0.1 mmol / L, and the mixture was induced at 18°C ​​and 200 r / min for 19 h. Cell pellets were collected by centrifugation at 4℃ and 5000g for 10 min. Samples before and after induction were analyzed by SDS-PAGE electrophoresis. The results are as follows: Figure 1 As shown, a significant target protein band appeared at approximately 12 kDa after induction, demonstrating successful expression of the nanobody.

[0018] Nanobody purification Periplasmic purification: The cell pellet was resuspended in 3.75 mL of pre-chilled TES buffer and incubated on an ice-cold rocker (100 rpm) for 1.5 h; 7.5 mL of TES / 4 buffer was added and incubated on an ice-cold rocker (100 rpm) for another 45 min; centrifuged at 12000 g for 30 min at 4 °C and the supernatant was collected. Nickel column affinity chromatography purification: The supernatant was loaded onto a nickel column, and impurities were washed with PBS buffer containing 20 mmol / L imidazole. The target protein was then eluted with PBS buffer containing 200 mmol / L imidazole. The elution peak was collected and dialyzed to obtain high-purity chlorantraniliprole nanobody.

[0019] Example 2: Detection of Nanobody Characteristics Sensitivity assays were performed by coating 96-well microplates with CYR-BSA diluted to 2 μg / mL using coating buffer and incubating at 37°C for 2 h. After BSA blocking, serial concentrations of CYR standards and 1:50 diluted nanobodies were added, and the reaction was carried out at 37°C for 1 h. HRP-labeled anti-His mouse monoclonal antibody was then added, and the plates were incubated at 37°C for 1 h. After TMB color development, the OD450 value was measured, and the IC50 value was calculated using Graphpad Prism 10.4.0 software. The results are shown below. Figure 2 As shown, the nanobody exhibits a half-maximal inhibitory concentration (WMC) of 1.04 μg / mL against chlorantraniliprole, demonstrating excellent sensitivity.

[0020] Organic solvent tolerance was tested by diluting nanobodies and CYR standards with PBS buffer containing 10%, 20%, 40%, 60%, and 80% methanol, acetonitrile, DMF, DMSO, and acetone. Antibody binding activity was then assessed, and the results are as follows: Figure 3 As shown: the activity of nanobody increases with increasing methanol concentration, has high tolerance to acetone (activity remains above 70%), retains more than 50% activity in 40% DMSO and DMF, and has poor tolerance to acetonitrile (activity decreases rapidly when concentration >10%).

[0021] Temperature tolerance testing was performed by incubating the nanobodies at 25, 50, 65, 80, and 95°C for 5 min, and at 90°C for 5, 15, 30, 45, 60, and 75 min, respectively. After returning to room temperature, antibody activity was measured. The results are as follows: Figure 4 As shown, the antibody activity remained above 80% after treatment at 90℃ for 30 minutes, indicating good thermal stability.

[0022] pH tolerance testing was performed by diluting the nanobody and CYR standard in a buffer solution with a pH range of 5.0–10.5. Antibody binding activity was measured, and inhibition curves were plotted. The results are as follows: Figure 5 As shown, the nanobody exhibits the lowest IC50 and highest activity at pH 7.4, and the inhibitory effect of alkaline conditions on antibody activity is greater than that of acidic conditions.

[0023] Ion strength tolerance was tested by diluting nanobodies and CYR standards with PBS buffer at ion concentrations of 5, 10, 25, 50, and 100 mmol / L. Antibody binding activity was measured and inhibition curves were plotted. The results are as follows: Figure 6 As shown, the nanobody exhibits the lowest IC50 value and the best activity at an ion concentration of 10 mmol / L, and its activity gradually decreases with increasing ion concentration.

[0024] Homology modeling and molecular docking analysis: The amino acid sequence of the nanobody was input into Discovery Studio 2019 software for homology modeling. The Ramachandran diagram analysis results are as follows: Figure 7 As shown, 95.2% of the atoms are distributed within a reasonable region, proving the excellent modeling results; the molecular docking results are as follows... Figure 8 , Figure 9 As shown, CYR molecules can be embedded in the catalytic cavity of nanobodies, forming hydrogen bonds with PRO108 and hydrophobic interactions with ALA99, PRO112, etc., resulting in strong binding stability.

[0025] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing chlorantraniliprole nanobodies, characterized in that, Includes the following steps: S1. Nanobody sequence screening: Specific nanobody sequences for chlorantraniliprole were obtained through screening using bioinformatics methods. S2. Expression vector construction: The screened nanobody gene was cloned into the pET-22b(+) expression vector to construct the pET22b-CYR nanobody inducible expression vector; S3. Strain culture and induction of expression: The pET22b-CYR expression vector was transformed into Escherichia coli BL21(DE3) competent cells to obtain the recombinant strain pET22b-CYR E.coli BL21(DE3), which was then cultured and induced to express by adding IPTG. S4. Nanobody Purification: Periplasmic purification combined with nickel column affinity chromatography was used to purify the recombinant nanobody with the His tag, obtaining high-purity chlorantraniliprole nanobody.

2. The method for preparing chlorantraniliprole nanobodies according to claim 1, characterized in that, The specific operations for bacterial culture and induced expression in step S3 include: The pET22b-CYR E.coli BL21(DE3) strain was streaked onto LB-Amp plates and incubated overnight at 37°C. Positive single colonies were selected and inoculated into LB liquid medium containing ampicillin. The culture was carried out at 37°C in a shaker until the OD600 value reached about 0.

6. IPTG was added to a final concentration of 0.1 mmol / L and induced at 18°C ​​for 18-20 h.

3. The method for preparing chlorantraniliprole nanobody according to claim 2, characterized in that, The specific operations of periplasmic purification in step S4 include: The induced cell pellet was resuspended in pre-cooled TES buffer and incubated on an ice-cold rocker for 1.5 h. Add TES / 4 buffer and continue incubation on an ice shaker for 45 min; centrifuge at 12000g for 30 min at 4℃ and collect the supernatant as peritoneal extract.

4. The method for preparing chlorantraniliprole nanobody according to claim 3, characterized in that, In step S4, nickel column affinity chromatography was used to purify the nanobodies in the periplasmic extract to obtain high-purity chlorantraniliprole nanobodies with His tags.

5. The method for preparing chlorantraniliprole nanobody according to claim 4, characterized in that: The nanobody exhibits a half-maximal inhibitory concentration (WMC) of 1.04 μg / mL against chlorantraniliprole, retains over 80% of its activity after treatment at 90°C for 30 min, and demonstrates good tolerance to organic solvents such as methanol and acetone.

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