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Article

Generation of an Insecticidal Human Domain Antibody from a Phage Library Targeting Plutella xylostella Brush-Border Membrane Vesicles

by
Xiaodan Hu
1,
Xiao Zhang
2,
Junmin Fan
1,
Wei Chen
2,
Fang Ji
1,
Qing Wang
1,
Juanjuan Tian
1,
Jinfeng Hou
1,
Hong Wang
3,
Shuting Yu
1,
Wenwen Qi
1,
Xianjin Liu
2,*,
Yuan Liu
2,* and
Chunqin Xie
1
1
Department of Tea and Food Science, Jiangsu Vocational College of Agriculture and Forestry, Zhenjiang 212400, China
2
Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
3
School of Pharmaceutical Engineering, Zhejiang Pharmaceutical University, Ningbo 315207, China
*
Authors to whom correspondence should be addressed.
Curr. Issues Mol. Biol. 2026, 48(2), 192; https://doi.org/10.3390/cimb48020192
Submission received: 8 January 2026 / Revised: 1 February 2026 / Accepted: 4 February 2026 / Published: 8 February 2026
(This article belongs to the Special Issue Molecular Dynamics Simulations in Structural Biology: From Proteins to Complex Biomolecular Systems)
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Abstract

The importance of protein-based materials in agricultural pest control has received increasing attention in recent years. Herein, Plutella xylostella brush-border membrane vesicles (BBMVs) were used as a target to screen for human domain antibodies with insecticidal activity. Three rounds of panning of the phage display library yielded the domain antibody C4D, which competed with the Cry1Ac toxin to bind to P. xylostella BBMVs. Against P. xylostella larvae, the recombinant soluble C4D protein showed an LC50 of 1.57 μg/cm2 (95% fiducial limits: 0.83–2.54). Using pull-down assays and liquid chromatography–tandem mass spectrometry, we identified the C4D binding partner in P. xylostella midgut BBMVs to be a cadherin-like protein. Bio-Layer Interferometry assay revealed that the dissociation constant between soluble C4D and P. xylostella cadherin-like protein was 2.99 × 10−6 M. Thus, the present study explored strategies to generate insecticidal antibodies, and the human domain antibody C4D identified and characterized in this study can serve as a framework for generating novel insecticidal agents.

1. Introduction

The diamondback moth Plutella xylostella (L.) (Lepidoptera: Plutellidae) is a destructive global lepidopteran pest [1]. The larvae of P. xylostella, equipped with chewing mouthparts, are voracious feeders that cause substantial damage to economically important cruciferous crops, including cabbage, cauliflower, broccoli, Brussels sprouts, Chinese cabbage, and Chinese kale [2,3]. Globally, annual economic losses due to P. xylostella amount to an estimated $4–5 billion [4]. Controlling the population of P. xylostella is challenging because of its remarkable environmental adaptability, high fecundity, and short generation time. The intensive, long-term use of chemical pesticides against P. xylostella has led to its resistance to 97 distinct chemical compounds, rendering it one of the most resistant insect species globally [5,6]. Therefore, alternative, eco-friendly control strategies are urgently needed.
Currently, Bacillus thuringiensis (Bt) and its insecticidal genes represent one of the most successful biotechnological tools in Integrated Pest Management, being both formulated into microbial insecticides and widely utilized in the development of transgenic insect-resistant crops. The wide application of Bt crystal proteins, known as Cry toxins, in pest management has led to a drastic reduction in insecticide use alongside significant economic, environmental, and social benefits [7,8]. The success of Cry toxins in pest control has established proteins as potential insecticidal agents. As per the immune network theory, specific antibodies (anti-idiotypic antibodies) can mimic the structure and function and act as copies of some antigens, including proteins, polypeptides, small molecules, and other substances [9,10]. Such specific antibodies have been widely used in biomedical applications, such as disease diagnosis, disease treatment, and vaccine development [11,12]. Given the broad and diverse functional capabilities of antibodies, such as in medicine and biotechnology, it can be hypothesized that certain antibodies exhibit insecticidal activity similar to that shown by Cry toxins. Single-domain antibodies (nanobodies) are the smallest antibody fragments with binding functionality [13]. They are considered a class of next-generation antibodies owing to their small size (approximately 2.5 × 4 nm), high tissue penetration capability, high conformational stability, and ease of production and engineering [14,15]. Moreover, human-derived antibodies exhibit lower immunogenicity than antibodies derived from other sources, such as mice, llamas, or rabbits, eliciting reduced immune responses in humans—a characteristic that enhances their safety profile as pesticides [13]. As insecticidal agents, human domain antibodies may offer distinct advantages: a safer profile from low human immunogenicity, ease of engineering due to their stable, simple structure, and scalable production. These advantages suggest that they represent a compelling novel strategy for environmentally friendly pest control.
Although the specific mechanism underlying the insecticidal activity of Cry proteins remains elusive, the general understanding is that they interact with receptor proteins in the brush-border membrane vesicles (BBMVs) of the insect midgut, ultimately inducing insect mortality [8,16,17]. Researchers have demonstrated marked enhancement in the insecticidal activity of Cry toxins by replacing some structural loops with peptides that can bind to BBMVs [18,19,20]. One of the primary mechanisms underlying insect resistance to Cry toxins involves alterations in the binding affinity of receptors on insect BBMVs to activated Bt toxins [21,22,23,24]. Given the integral role of the insect midgut in digestion and nutrient absorption, BBMVs represent a promising target for insecticide development.
Phage display technology, which was awarded the Nobel Prize in Chemistry in 2018, serves as a powerful and versatile method, allowing for the discovery of proteins with both high affinity and specificity [25]. It is based on the in vitro selection of peptides or proteins displayed as fusion proteins on the surface of a bacteriophage [26]. In addition to its extensive use in several clinical and therapeutic applications, this technology is increasingly being used in the fields of food safety and pest control [27,28,29].
In the present study, P. xylostella BBMVs were used as antigens to screen for human domain antibodies with insecticidal activity. After three rounds of panning, an antibody called C4D was isolated from a human domain antibody library. C4D was found to compete with Cry1Ac toxin for binding to P. xylostella BBMVs. C4D was recombinantly expressed in Escherichia coli BL21 (DE3) cells, and the insecticidal activity of soluble C4D against P. xylostella was demonstrated through bioassays. The specific BBMVs protein targeted by C4D was isolated using a pull-down assay and identified using liquid chromatography–tandem mass spectrometry (LC–MS/MS) as P. xylostella cadherin-like protein.

2. Materials and Methods

2.1. Amplification of Phage Human Domain Antibodies and Extraction of Brush-Border Membrane Vesicles

The domain antibody library, obtained from the Medical Research Council HGMP Resource Center, contains 3 × 109 phages, with each phage displaying a different antibody fragment fused to its terminal phage geneIII protein. The domain antibodies were derived from a single human VH framework (V3–23/D47), with diversity introduced only in the antigen-binding site, which comprises complementarity-determining region 1 (CDR1), CDR2, and CDR3. The phage antibody repertoire of the naive domain antibody library was produced by infecting the stocked E. coli TG1 library with KM13 helper phages following a standard protocol [30].
A total of 500 fourth-instar P. xylostella larvae of the HN strain, starved for 3 h, were aseptically dissected under a microscope and immediately subjected to a wash with ice-cold 150 mM NaCl solution. The midguts were transferred to a sterile homogenizer containing 3 mL of homogenization buffer (300 mM mannitol, 5 mM EGTA, 17 mM Tris, pH 7.5), homogenized for 30 s, and incubated on ice for 30 s. This cycle was repeated six times until no visible tissue fragments remained. The resulting homogenate was transferred to a 50 mL centrifuge tube. Subsequently, an equal volume of ice-cold 24 mM MgCl2 solution was added. The mixture was incubated on ice for 30 min, followed by centrifugation at 3500× g for 15 min at 4 °C. The supernatant was carefully collected in a new microcentrifuge tube and centrifuged at 30,000× g for 1 h at 4 °C. The pellet was carefully isolated and resuspended in 500 μL of HEPES buffer (10 mM HEPES, 130 mM KCl, 10% glycerol, pH 7.4). The resultant BBMVs’ suspension was aliquoted and stored at −80 °C.

2.2. Human Domain Antibody Library Panning and Identification of Domain Antibodies Targeting P. xylostella BBMVs

To enrich the specific domain antibodies targeting P. xylostella BBMVs, the rescued phage domain antibody library was subjected to panning (Figure 1). Briefly, a well of a high-protein-binding plate was coated with 1 mL of a 100 μg/mL BBMVs solution prepared in phosphate-buffered saline (PBS; 0.2 g KCl, 3.6 g Na2HPO4,8 g NaCl, and 0.24 g KH2PO4 dissolved in 1 L of deionized water, pH 7.4) overnight at 4 °C. The next day, the well was subjected to four washes with 5 mL of PBS, and the unoccupied sites were blocked with 3 mL of PBS containing 5% (w/v) skim milk (MPBS) at 37 °C for 2 h. The well was washed four times again with PBS, followed by the addition of the phage domain antibody library, which contained 2.3 × 1011 phages in 1 mL of 5% (w/v) MPBS. The plate was gently agitated at 100 rpm for 1 h and allowed to stand for another 1 h at 25 °C. The phages were removed. The well was first washed ten times with 5 mL of PBST (PBS with 0.1% Tween-20). This was followed by two additional washes with 5 mL of PBS. Unbound or weakly bound phage particles were first washed away. Subsequently, the remaining bound phages were eluted by incubation with 1 mL of 150 μg/mL trypsin protease (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature with gentle agitation at 100 rpm. Eluted phages were mixed with log-phase E. coli TG1 cultures and incubated for 1 h at 37 °C. For isolation of E. coli TG1 clones harboring antibody genes, the mixture was plated on tryptone-yeast extract plates containing 100 μg/mL ampicillin. The clones obtained were used to titer the output phage and amplify phage antibodies for the next round of panning. In the next two rounds of panning, trypsin was replaced with 200 μg/mL Cry1Ac toxins to elute the specific binders, and a reduced BBMV coating concentration and more stringent washing steps were implemented to enhance the enrichment of high-affinity binders. The second round of panning featured a 50 μg/mL solution of BBMVs and 15 washes with PBST, while the third round featured a 25 μg/mL solution of BBMVs and 20 washes with PBST.

2.3. Phage Enzyme-Linked Immunosorbent Assay

The polyclonal phages obtained after each round of panning were tested by phage enzyme-linked immunosorbent assay (ELISA). The output phages of each round were amplified, and 1 × 109 phages were added to wells that had been coated with 100 μL of 30 μg/mL BBMVs in 5% (w/v) solution and blocked with 300 μL of 5% (w/v) MPBS. After being incubated at 37 °C for 1 h, the wells were subjected to four washes with PBST. To detect bound phages, 100 μL of horseradish peroxidase (HRP)-conjugated anti-M13 monoclonal antibody (1:3000; Sino Biological Inc., Beijing, China) was added to the wells and incubated at 37 °C for 1 h. After washing the wells four times with PBST, 100 μL of 3,3′,5,5′-tetramethylbenzidine substrate (GenScript, Piscataway, NJ, USA) was added to each well and incubated at 37 °C for 15 min. The reaction was stopped by adding 50 μL of 2 M sulfuric acid. Subsequently, the absorbance at 450 nm was recorded using a UV-Vis plate reader. Wells coated with 5% (w/v) MPBS were defined as the negative control. The phage pools with the highest absorbance values were chosen for monoclonal phage ELISA. Three independent dilution processes were performed, with phages from each process assayed in technical triplicate.
Following random selection, individual clones from the output library were rescued with KM13 helper phage. The produced monoclonal phage antibodies were then analyzed by ELISA against BBMVs. The protocol for monoclonal phage ELISA was the same as that for polyclonal phage ELISA. Clones with specific BBMVs binding were selected for further analysis. Three independent rescue processes were performed, with phages from each process assayed in technical triplicate.
Antibodies showing the highest enrichment and strongest binding to BBMVs were selected for competitive ELISA, which was performed in the same manner as monoclonal phage ELISA, except that the monoclonal phages were incubated with 100 μg/mL Cry1Ac. Three independent rescue processes were performed, with phages from each process assayed in technical triplicate.
The ELISA results were analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test, using GraphPad Prism 10. Data are from three independent experiments (n = 3).

2.4. Expression and Purification of Soluble C4D

To analyze the insecticidal activity of human domain antibody C4D, soluble C4D protein was expressed using a prokaryotic expression system. The coding sequence of human domain antibody C4D was subjected to amplification with the primers C4D-F (5′-CATGCCATGGCCCAGGTGCAGCTGTTGGAGTCTG-3′), which contained a NcoI site (italicized), and C4D-R (5′-ATAAGAATGCGGCCGCCAGATCCTCTTCAGAGATGAGTTTCTGCTCGCTCGAGACGGTGAC-3′), which contained a NotI site (italicized) and a c-Myc label. The amplicon was cloned into pET26b (+) vector (Novagen, Madison, WI, USA), and the recombinant plasmid was transformed into E. coli BL21 (DE3) cells (TransGen Biotech, Beijing, China) to express soluble C4D protein. Following induction with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG; Invitrogen, Carlsbad, CA, USA), the bacterial culture was cultivated for 16 h at 25 °C with agitation (220 rpm). The induced cells were harvested and lysed by sonication. Following centrifugation of the lysates, the clarified supernatants were subjected to purification using a nickel–nitrilotriacetic acid (Ni-NTA) affinity column (GE Healthcare, Piscataway, NJ, USA) to isolate soluble C4D. The purified protein was desalted using a desalting column (GE Healthcare, Piscataway, NJ, USA). The concentration of purified C4D was measured using a Nanodrop spectrophotometer. Subsequently, the protein was aliquoted and stored at −80 °C.

2.5. Insect Bioassays

For insect bioassays, plastic Petri dishes (6 cm in diameter) were coated with insect diet. Each insect diet was overlaid with 1 mL of C4D protein solution (at varying concentrations in PBS) to achieve final concentrations of 0.4, 0.8, 1.6, 3.2, 6.4 or 12.8 μg/cm2 and was then allowed to air-dry. For each treatment, five plates (replicates) were prepared, with 20 s-instar P. xylostella larvae of the HN strain were placed on each dried diet surface, resulting in a total of 100 larvae per treatment. The D3H clone, which was obtained from the same human domain antibody library panning but showed no competitive activity with Cry1Ac, and PBS were used as negative control. A series of concentrations of Cry1Ac toxin was used as the positive control. Mortality was assessed after 5 days, and larvae that failed to pupate were also recorded as dead. The concentration of proteins that kills 50% of the larvae (LC50) was calculated using GraphPad Prism 10 software.

2.6. Pull-Down Assays

The P. xylostella midgut BBMVs proteins interacting with C4D were captured through pull-down assays performed using Dynabeads™ His-Tag isolation and pull-down beads (Invitrogen, Carlsbad, CA, USA). Briefly, 50 μL of a resuspension of Dynabeads™ magnetic beads was transferred to a microcentrifuge tube. After a brief (2 min) magnetic separation, the supernatant was aspirated and discarded. Next, 700 μL of 1.5 mg/mL soluble C4D protein dissolved in 1 × binding/wash buffer (300 mM NaCl, 50 mM sodium phosphate, 0.01% Tween-20, pH 8.0) was added to the beads and mixed well. The tube was subjected to incubation for 15 min at 25 °C on a roller, and then placed on a magnet for 2 min, following which the supernatant was discarded. The beads underwent four washes, each using 300 μL of 1 × Binding/Wash Buffer, and the supernatant was discarded after every wash. Next, 700 μL of 1 mg/mL BBMVs dissolved in 1 × pull-down buffer (3.25 mM sodium phosphate, 70 mM NaCl, 0.0% Tween-20, pH 7.4) was added to the beads. The tube was incubated on a roller for 30 min at 4 °C and then placed on a magnet for 2 min, following which the supernatant was discarded. After washing the beads four times with 300 μL of 1 × Binding/Wash Buffer and discarding the supernatant, 50 μL of His-elution buffer (300 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, 0.01% Tween-20, pH 8.0) was added to the tube. The tube was placed on a roller for 5 min at 4 °C. The beads were collected using a magnet, and the supernatant—containing the interaction partner of C4D—was transferred to a fresh tube. The beads not incubated with C4D served as a control. The isolated protein was first analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The protein band was excised from the gel and further analyzed via LC–MS/MS. The analysis was performed on a Thermo Fisher Q Exactive Plus system equipped with a Nano Flex ion source operating in data-dependent acquisition mode. The raw mass spectrometry data were processed using Proteome Discoverer 2.5 software and subsequently searched against the UniProt Plutella xylostella database with a false discovery rate (FDR) threshold set to <1% at all identification levels.

2.7. Bio-Layer Interferometry Assay (BLI)

The binding between soluble C4D and P. xylostella cadherin-like fragment (amino acids T1202 to I1447) was analyzed by BLI using the Octet RED96e system (Sartorius, Goettingen, Germany). Plutella xylostella cadherin-like fragment was first expressed recombinantly in our laboratory [31]. The BLI was performed using aminopropylsilane (APS)-coated biosensor tips (Sartorius Goettingen, Germany). First, tips were immersed in a solution containing P. xylostella cadherin-like fragment at a concentration of 20 μg/mL for 3 min to allow for immobilization. Following this, the tips were immersed in kinetic buffer (PBS supplemented with 0.1% (w/v) bovine serum albumin, BSA) for 30 s. For affinity measurement, soluble C4D was serially diluted in kinetic buffer to a series of concentrations (1 μM, 0.5 μM, 0.25 μM, 0.125 μM), which were then applied to the immobilized tips to monitor binding interactions. All binding signals were recorded using the Octet System Data Acquisition software (version 9.0, Sartorius, Goettingen, Germany). The soluble D3H was used as negative control.

3. Results

3.1. Enrichment of Domain Antibodies Binding to Brush-Border Membrane Vesicles

The magnitude of enrichment can be estimated by calculating the proportion of input phages that bind to BBMVs. The input phage number for each round of panning was maintained at 1011, while the relative proportion of output phage increased from the first to the third round, confirming the successful enrichment of domain antibodies targeting BBMVs (Table 1).

3.2. ELISA Analysis of Phage Display Screening

Phages that became amplified after each round of selection were diluted to 1 × 1010 colony-forming units/mL and subjected to polyclonal phage ELISA. The absorbance for BBMVs binding increased significantly with each panning round, with the signal after the third round being significantly higher than after the first (p < 0.0001) and second rounds (p < 0.0001), as shown in Figure 2A. In contrast, the binding signal to the MPBS control remained low and was significantly lower (p = 0.0021) than that of the naïve phage library (Figure 2A). These results indicate the successive enrichment of BBMVs-specific binders.
A total of 380 individual clones were randomly selected from the output of the third round of panning and subjected to monoclonal ELISA against BBMVs. MPBS served as a control. Clones with ELISA signals at least 3-fold higher in intensity than the control were selected as positive binders to BBMVs. As a result, 32 positive clones were selected and sequenced.
Sequencing results revealed several identical antibody sequences. We selected the six most frequently occurring sequences for subsequent experiments. The representative strains corresponding to each sequence were A5E, A7C, B4C, C4D, D2F, and D3H, and the number of clones with identical amino acid sequences as these strains was 3, 6, 5, 5, 3, and 4, respectively. The results of monoclonal ELISA are shown in Figure 2B.
The six representative clones selected from monoclonal phage ELISA were subjected to competitive ELISA to assess whether they competed with Cry1Ac toxin in binding to BBMVs. Competitive ELISA (Figure 2C) revealed that among the six domain antibodies tested, only the binding of C4D to BBMVs was significantly inhibited by Cry1Ac toxin, decreasing by 45.95% (from 0.75 ± 0.02 to 0.40 ± 0.02; p < 0.0001). In contrast, the binding of the other five antibodies was not significantly affected (all p > 0.05).

3.3. Expression and Purification of Soluble C4D

The recombinant E. coli BL21 (DE3) clone was sequenced to verify the correct insertion of the C4D antibody gene into pET-26b (+) vector. The expression of soluble C4D protein was verified by SDS–PAGE and Western blotting (Figure 3). The amino acid sequence of human domain antibody C4D is presented in Figure 3A. E. coli BL21 (DE3) cells harboring empty pET-26b (+) vector and induced at the same time were used as a control. The results of SDS–PAGE analysis showed that E. coli BL21 (DE3) cells harboring the C4D coding sequence exhibited a specific protein band at approximately 15 kDa (Figure 3B), the same position at which a sharp band was observed when the purified protein was run on SDS–PAGE. Given that C4D was expressed with a c-Myc fusion tag, the purified soluble C4D protein was detected by Western blotting with an HRP-conjugated c-Myc antibody (GenScript, Piscataway, NJ, USA). The Western blotting results showed a specific band at the same position as the band on SDS–PAGE, and the molecular weight was consistent with that of soluble C4D (Figure 3C). These results verified the successful expression and purification of soluble C4D, and SDS-PAGE analysis also demonstrated high purity of the recombinant C4D protein (Figure 3B). The purified protein was obtained at a yield of approximately 1.5 mg/mL in PBS and stored at −80 °C.

3.4. Insect Bioassay

In the insect bioassays, the LC50 of Cry1Ac was 6.60 ng/cm2 (95% fiducial limit: 6.10–7.14), suggesting these insects were sensitive to Cry1Ac and could be used for bioassays. The results of the insect bioassay revealed the LC50 of C4D was 1.57 μg/cm2 (95% fiducial limit: 0.83–2.54), whereas no mortality was observed in the D3H group at the concentration of 20 μg/cm2, confirming the insecticidal activity of C4D antibody (Table 2).

3.5. Pull-Down Assays

The protein isolated through pull-down assays was first analyzed by SDS–PAGE. Compared with the control, the pull-down sample displayed a specific band at approximately 180 kDa (Figure 4A). The band was excised, trypsin-digested, and analyzed by LC–MS/MS (Figure 4B). The analysis identified the target protein as the P. xylostella cadherin-like protein (GenBank: AAS98023.1). Under stringent filtering criteria, this identification was supported by 197 high-confidence peptide-spectrum matches (PSMs), corresponding to 26 unique peptides and yielding a sequence coverage of 13%. The mass spectrum of one peptide is shown in Figure 5B. Plutella xylostella cadherin-like protein was previously reported to be a protein present in P. xylostella BBMVs, functioning as a Cry1Ac receptor [31].

3.6. Binding Activity Analysis of Soluble C4D Protein with P. xylostella Cadherin-like Fragment

BLI analysis was performed to assess binding. The results revealed that while C4D could bind to the P. xylostella cadherin-like fragment, D3H showed no binding activity (Figure 5A). BLI analysis demonstrated a concentration-dependent increase in the binding response of soluble C4D to the P. xylostella cadherin-like fragment, confirming their specific interaction. The measured binding affinity (KD) was 2.99 × 10−6 M, with an association rate constant (Ka) of 1.28 × 103 M−1s−1and a dissociation rate constant (Kb) of 3.83 × 10−3 s−1 (Figure 5B).

4. Discussion

Bt toxins, which have been successfully formulated into microbial insecticides and engineered into transgenic crops, represent a cornerstone of modern insect control and are the most extensively studied insecticidal proteins to date. According to the commonly accepted mechanism, Cry toxins exert their insecticidal activity by binding to receptors on the surface of BBMVs in the insect midgut [8,16,32]. Shao et al. demonstrated a 9-fold increase in the toxicity of Cry1Ab toward the brown planthopper Nilaparvata lugens by replacing the domain II loops of the toxin with BBMVs-binding peptides [19]. Similarly, Chougule et al. enhanced the toxicity of Cyt2Aa toward the pea aphid Acyrthosiphon pisum and the green peach aphid Myzus persicae by replacing one loop of the toxin with BBMVs-binding peptides [33]. Given the integral role of the insect midgut in digestion and nutrient absorption, BBMVs represent a promising target for insecticide development. Phage display technology is an efficient, high-throughput method for high-affinity and high-specificity antibody discovery. During panning, antigen-specific competitive elution is an efficient strategy to facilitate target antibody isolation, proving advantageous for the production of anti-idiotypic antibody fragments [34]. To maximize the isolation of human domain antibodies with insecticidal activity against P. xylostella, we conducted competitive panning of phage-displayed libraries on BBMVs substrates using Cry1Ac as the displacement eluent. In Round 1 of screening, trypsin elution was employed to maximize recovery of all BBMVs-bound antibodies, followed by amplification to expand the population for subsequent selection—a strategy that prevented the loss of binding clones under stringent initial conditions. Polyclonal analysis confirmed that BBMVs-binding antibodies were effectively enriched after Round 1. In Round 2, competitive elution was performed with Cry1Ac toxin, and no significant increase was noted post-Round 2 compared with Round 1. This was potentially due to the displacement of antibodies binding to BBMVs sites not targeted by Cry1Ac. Competitive screening further enriched target-specific antibodies in Round 3, in alignment with the planned selection strategy. The predominance of noncompetitively eluted antibodies after screening may be a result of the high fluid shear stress generated during microfluidic washing, causing the dissociation of lower-affinity antibodies against BBMVs.
Panning of the human antibody library yielded the insecticidal domain antibody C4D, which competed with the Cry1Ac toxin to bind to P. xylostella BBMVs. The pull-down assays, LC–MS/MS, and BLI analysis identified and verified the C4D binding partner in P. xylostella midgut BBMVs to be midgut cadherin-like protein. In invertebrates, cadherin-like protein belongs to a major family of calcium-dependent transmembrane glycoproteins. Their critical functions encompass the regulation of cell migration, intercellular adhesion, tissue organization, and morphogenesis [35]. Cadherin-like proteins also engage in signal transduction. Their ectodomain mediates interactions with other adhesion molecules, and their cytoplasmic domain connects to specific intracellular proteins, facilitating downstream signaling [36]. Current research on insect midgut cadherin-like proteins primarily focuses on their role in the mode of action of Cry toxins. Although Cry toxins target multiple receptors and their precise mechanism of toxicity remains unclear, cadherin-like proteins in various insect species have been confirmed as one of the functional receptors for Cry toxins [37,38,39]. Gao et al. investigated the binding of Cry toxin to P. xylostella cadherin-like protein and the subsequent cellular toxicity, concluding that P. xylostella cadherin-like protein might function as a Cry1Ac receptor [31]. Crucially, in one of the widely accepted insecticidal mechanisms of Cry1A toxins, insect cadherin-like protein can activate an intracellular death-signaling pathway analogous to that of GPCRs. Owing to this functional homology coupled with structural similarities, some researchers classified it within the cadherin-GPCR family [40,41]. GPCRs have long been established as valuable targets in human drug discovery and are now also considered for pest management. In recent years, insect GPCRs have emerged as particularly promising targets for next-generation pesticides. Their high species specificity further offers the potential for selective pest control with minimal off-target effects [42]. Therefore, our findings demonstrate a correlation between C4D binding to cadherin-like proteins and larval mortality, but do not elucidate the downstream toxicological mechanism. The precise mechanism by which this binding leads to death remains speculative and constitutes an important objective for future investigation.
In the present research, we obtained a human domain antibody, C4D, with insecticidal activity. Xie et al. previously reported an antibody called GEAb-dVL, consisting of two light chains, that demonstrated insecticidal activity against P. xylostella larvae [43]. Lin et al. identified a single-chain variable fragment antibody called M4 that induced mortality against P. xylostella larvae [44]. All these studies confirm that insecticidal activity can be attributed to some specific antibodies. However, when quantitatively benchmarked against established toxins, the current efficacy of such antibody-based agents remains substantially lower. The Cry1Ac toxin, for example, exhibits an LC50 of 6.6 ng/cm2 against P. xylostella. In comparison, the human single-domain antibody C4D reported here has an LC50 of approximately 1.57 µg/cm2–about 240-fold higher than that of Cry1Ac. This gap in potency is further contextualized by other antibody formats: GEAb-dVL shows an LC50 of 235 µg/cm2, and M4 induces only 27.8% mortality at 53 µg/cm2 [43,44]. Therefore, while C4D represents the most active human antibody-derived scaffold identified in our study, its activity level clearly positions it as a proof-of-concept molecule rather than a field-ready biopesticide. These results highlight that the primary value of C4D is in demonstrating the feasibility of engineering human domain antibodies for insecticidal function. This represents a foundational step toward designing future biocontrol agents with improved efficacy and modularity for pest management. In Cry toxins, the α-helical conformation is central to the pore-forming ability of the toxin, and hence, to its toxicity. Surprisingly, antibodies lacking this secondary structure element retained their insecticidal activity. Previous studies have implicated the surface-exposed loops of Cry toxins in receptor interactions [45]. Interestingly, antibodies interact with specific antigens through CDRs on the apex [46]. In addition to their similarity in position and role in interacting with binding partners, both Cry toxin loops and CDRs of antibodies display marked diversity in their sequence composition, length, and structural conformations [46,47]. These functional and structural similarities between Cry toxin loops and CDRs of antibodies may be the reason why antibodies can show insecticidal activity. The human domain antibody C4D obtained in this study exhibited approximately 240-fold higher LC50 than Cry1Ac against P. xylostella larvae, probably because of its low binding affinity to P. xylostella cadherin-like protein and its simple structure compared with Cry1Ac. Our future research will aim to enhance the binding affinity of C4D for cadherin-like protein through antibody evolution and reinforce structural stability through multimerization engineering, thereby boosting insecticidal activity.

5. Conclusions

Herein, P. xylostella BBMVs were used as a target to screen for human domain antibodies with insecticidal activity. After three rounds of panning, we isolated C4D, a human domain antibody that could compete with Cry1Ac toxin to bind to P. xylostella BBMVs. Recombinant soluble C4D was expressed in E. coli BL21 (DE3) cells, and bioassays were performed to validate the insecticidal activity of soluble C4D against P. xylostella. The specific binding partner of C4D was isolated using pull-down assays and identified using LC–MS/MS as P. xylostella cadherin-like protein, a functional Cry toxin receptor. The binding between soluble C4D and P. xylostella cadherin-like protein was verified through BLI analysis. Thus, the present study explored strategies to generate insecticidal antibodies, laying the groundwork for the generation of new insecticidal agents.

Author Contributions

Conceptualization, X.H. and X.L.; methodology, X.H. and X.Z.; software, J.F.; validation, F.J., S.Y. and W.Q.; formal analysis, H.W. and J.H.; investigation, X.H., W.C., Q.W. and J.T.; resources, X.H. and Y.L.; data curation, X.Z.; writing—original draft preparation, X.H.; writing—review and editing, X.H., Y.L. and X.Z.; supervision, C.X. and X.L.; project administration, C.X.; funding acquisition, X.H., X.L. and F.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by General Program of Natural Science Foundation of Jiangsu Provincial Institutions of Higher Education (24KJB210010), Scientific Research Startup Fund for High-Level Talents of Jiangsu Vocational College of Agriculture and Forestry (2024rc39), Jiangsu Province Agricultural Science and Technology Independent Innovation Fund [CX(22)1009], General Program of Jiangsu Provincial Natural Science Foundation (BK20231248) and Science and Technology Project for Social Development of Jurong (ZA42316).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author(s).

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
Bt Bacillus thuringiensis
BBMVsBrush-border membrane vesicles
LC–MS/MSLiquid chromatography–tandem mass spectrometry
CDRComplementarity-determining region
PBSPhosphate-buffered saline
MPBSSkim milk dissolved in phosphate-buffered saline
PBSTPhosphate-buffered saline containing 0.1% Tween-20
ELISAEnzyme-linked immunosorbent assay
HRPHorseradish peroxidase
SDS–PAGESodium dodecyl sulfate–polyacrylamide gel electrophoresis

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Cimb 48 00192 g001
Figure 1. Schematic of the panning process for selecting BBMVs-targeted human domain antibodies. The procedure consisted of the following key steps: (1–2) Pre-clearance by removing phages binding to 5% (w/v) MPBS; (3–4) Washing to remove non-specifically bound phages; (5) Elution of phages specifically bound to BBMVs; (6) Amplification of the eluted phage pool for subsequent rounds of selection or analysis.
Figure 1. Schematic of the panning process for selecting BBMVs-targeted human domain antibodies. The procedure consisted of the following key steps: (1–2) Pre-clearance by removing phages binding to 5% (w/v) MPBS; (3–4) Washing to remove non-specifically bound phages; (5) Elution of phages specifically bound to BBMVs; (6) Amplification of the eluted phage pool for subsequent rounds of selection or analysis.
Cimb 48 00192 g001
Cimb 48 00192 g002
Figure 2. ELISA analysis of phage display screening. (A) ELISA analysis of polyclonal phages rescued after each round of screening. (B) Monoclonal phage ELISA analysis of third-round output clones. Each clone was rescued with KM13 helper phage and adjusted to an equivalent titer prior to analysis. (C) The competition was observed in the presence of 100 μg/mL Cry1Ac. Differences in ELISA results were assessed by one-way ANOVA followed by Tukey’s multiple comparisons test, using GraphPad Prism 10. The data are presented as the mean values ± SD. Different letters indicate significant differences between groups at p < 0.05. (n = 3).
Figure 2. ELISA analysis of phage display screening. (A) ELISA analysis of polyclonal phages rescued after each round of screening. (B) Monoclonal phage ELISA analysis of third-round output clones. Each clone was rescued with KM13 helper phage and adjusted to an equivalent titer prior to analysis. (C) The competition was observed in the presence of 100 μg/mL Cry1Ac. Differences in ELISA results were assessed by one-way ANOVA followed by Tukey’s multiple comparisons test, using GraphPad Prism 10. The data are presented as the mean values ± SD. Different letters indicate significant differences between groups at p < 0.05. (n = 3).
Cimb 48 00192 g002
Cimb 48 00192 g003
Figure 3. Expression and analysis of the soluble human domain antibody C4D. (A) Amino acid sequence of human domain antibody C4D and D3H. Lane M, protein marker; Lane 1, E. coli BL21 (DE3) harboring empty vector; Lane 2, Purified soluble C4D protein; Lane 3, E. coli BL21 (DE3) harboring pET-26b (+)-C4D. (B) Analysis of C4D expression by SDS-PAGE. (C) Western blotting analysis of soluble C4D expression with c-Myc antibodies.
Figure 3. Expression and analysis of the soluble human domain antibody C4D. (A) Amino acid sequence of human domain antibody C4D and D3H. Lane M, protein marker; Lane 1, E. coli BL21 (DE3) harboring empty vector; Lane 2, Purified soluble C4D protein; Lane 3, E. coli BL21 (DE3) harboring pET-26b (+)-C4D. (B) Analysis of C4D expression by SDS-PAGE. (C) Western blotting analysis of soluble C4D expression with c-Myc antibodies.
Cimb 48 00192 g003
Cimb 48 00192 g004
Figure 4. Pull-down assay identifying C4D-binding proteins from P. xylostella BBMVs. (A) SDS-PAGE of eluted proteins. Lane M: Marker; Lane 1: Beads + BBMVs (control); Lane 2: Beads + C4D + BBMVs. (B) Representative LC-MS/MS spectrum confirming P. xylostella cadherin-like protein. The peptide sequence [R].IVSDISESFKVDAEKNIISLTR.[E] was matched to P. xylostella cadherin-like protein. Peaks correspond to fragment ions, with m/z and relative intensity shown on the x- and y-axes, respectively.
Figure 4. Pull-down assay identifying C4D-binding proteins from P. xylostella BBMVs. (A) SDS-PAGE of eluted proteins. Lane M: Marker; Lane 1: Beads + BBMVs (control); Lane 2: Beads + C4D + BBMVs. (B) Representative LC-MS/MS spectrum confirming P. xylostella cadherin-like protein. The peptide sequence [R].IVSDISESFKVDAEKNIISLTR.[E] was matched to P. xylostella cadherin-like protein. Peaks correspond to fragment ions, with m/z and relative intensity shown on the x- and y-axes, respectively.
Cimb 48 00192 g004
Cimb 48 00192 g005
Figure 5. BLI analysis of human domain antibodies binding to the P. xylostella cadherin-like fragment. (A) BLI analysis of different antibodies binding to the P. xylostella cadherin-like fragment. (B) BLI analysis of C4D binding to the P. xylostella cadherin-like fragment. The binding affinity was measured using an Octet RED96e system.
Figure 5. BLI analysis of human domain antibodies binding to the P. xylostella cadherin-like fragment. (A) BLI analysis of different antibodies binding to the P. xylostella cadherin-like fragment. (B) BLI analysis of C4D binding to the P. xylostella cadherin-like fragment. The binding affinity was measured using an Octet RED96e system.
Cimb 48 00192 g005
Table 1. Recovery of phages during selection against BBMVs.
Table 1. Recovery of phages during selection against BBMVs.
Selection RoundInput aOutput a,bPhage Bound c
12.3 × 10111.2 × 1045.2 × 10−8
22.0 × 10111.2 × 1056.0 × 10−7
31.5 × 10111.9 × 1071.3 × 10−4
a Phage inputs and outputs were calculated by transduction to E. coli TG1. b Number of eluted phages expressed as colony-forming unit per milliliter (cfu/mL). c Phage bound = output/input.
Table 2. Toxicity of the tested proteins against P. xylostella.
Table 2. Toxicity of the tested proteins against P. xylostella.
TreatmentLC50
(95% Fiducial Limits) (ng/cm2)		R2HillSlopeDegrees of Freedom
Cry1Ac6.60 (6.10–7.14)0.99−1.413
C4D1.57 (0.83–2.54) × 1030.87−0.525
D3H>20,000 a
a Plutella xylostella showed no mortality at the maximum concentration tested (20,000 ng/cm2).
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MDPI and ACS Style

Hu, X.; Zhang, X.; Fan, J.; Chen, W.; Ji, F.; Wang, Q.; Tian, J.; Hou, J.; Wang, H.; Yu, S.; et al. Generation of an Insecticidal Human Domain Antibody from a Phage Library Targeting Plutella xylostella Brush-Border Membrane Vesicles. Curr. Issues Mol. Biol. 2026, 48, 192. https://doi.org/10.3390/cimb48020192

AMA Style

Hu X, Zhang X, Fan J, Chen W, Ji F, Wang Q, Tian J, Hou J, Wang H, Yu S, et al. Generation of an Insecticidal Human Domain Antibody from a Phage Library Targeting Plutella xylostella Brush-Border Membrane Vesicles. Current Issues in Molecular Biology. 2026; 48(2):192. https://doi.org/10.3390/cimb48020192

Chicago/Turabian Style

Hu, Xiaodan, Xiao Zhang, Junmin Fan, Wei Chen, Fang Ji, Qing Wang, Juanjuan Tian, Jinfeng Hou, Hong Wang, Shuting Yu, and et al. 2026. "Generation of an Insecticidal Human Domain Antibody from a Phage Library Targeting Plutella xylostella Brush-Border Membrane Vesicles" Current Issues in Molecular Biology 48, no. 2: 192. https://doi.org/10.3390/cimb48020192

APA Style

Hu, X., Zhang, X., Fan, J., Chen, W., Ji, F., Wang, Q., Tian, J., Hou, J., Wang, H., Yu, S., Qi, W., Liu, X., Liu, Y., & Xie, C. (2026). Generation of an Insecticidal Human Domain Antibody from a Phage Library Targeting Plutella xylostella Brush-Border Membrane Vesicles. Current Issues in Molecular Biology, 48(2), 192. https://doi.org/10.3390/cimb48020192

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