Rational design and application of broad-spectrum antibodies for Bt Cry toxins determination.

Using the amino acid sequences and analysis of selected known structures of Bt Cry toxins, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F we specifically designed immunogens. After antibodies selection, broad-spectrum polyclonal antibodies (pAbs) and monoclonal antibody (namely 1A0-mAb) were obtained from rabbit and mouse, respectively. The produced pAbs displayed broad spectrum activity by recognizing Cry1 toxin, Cry2Aa, Cry2Ab and Cry3Aa with half maximal inhibitory concentration (IC50) values of 0.12-9.86 µg/mL. Similarly, 1A0-mAb showed broad spectrum activity, recognizing all of the above Cry protein (IC50 values of 4.66-20.46 µg/mL) with the exception of Cry2Aa. Using optimizations studies, 1A10-mAb was used as a capture antibody and pAbs as detection antibody. Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) were established for Cry1 toxin, Cry2Ab and Cry3Aa with the limit of detection (LOD) values of 2.36-36.37 ng/mL, respectively. The present DAS-ELISAs had good accuracy and precisions for the determination of Cry toxin spiked tap water, corn, rice, soybeans and soil samples. In conclusion, the present study has successfully obtained broad-spectrum pAbs and mAb. Furthermore, the generated pAbs- and mAb-based DAS-ELISAs protocol can potentially be used for the broad-spectrum monitoring of eight common subtypes of Bt Cry toxins residues in food and environmental samples.

Characterization of the individual domains of the Bacillus thuringiensis Cry2Aa implicates Domain I as a possible binding site to Helicoverpa armigera.

Bacillus thuringiensis (Bt) Cry2Aa is a member of the Cry pore-forming, 3-domain, toxin family with activity against both lepidopteran and dipteran insects. Although domains II and III of the Cry toxins are believed to represent the primary specificity determinant through specific binding to cell receptors, it has been proposed that the pore-forming domain I of Cry2Aa also has such a role. Thus, a greater understanding of the functions of Cry2Aa's different domains could potentially be helpful in the rational design of improved toxins. In this work, cry2Aa and its domain fragments (DI, DII, DIII, DI-II and DII-DIII) were subcloned into the vector pGEX-6P-1 and expressed in Escherichia coli. Each protein was recognized by anti-Cry2Aa antibodies and, except for the DII fragment, could block binding of the antibody to Cry2Aa. Cry2Aa and its DI and DI-II fragments bound to brush border membrane vesicles (BBMV) from H. armigera and also to a ca 150 kDa BBMV protein on a far western (ligand) blot. In contrast the DII, DIII and DII-III fragments bound to neither of these. None of the fragments were stable in H. armigera gut juice nor showed any toxicity towards this insect. Our results indicate that contrary to the general model of Cry toxin activity domain I plays a role in the binding of the toxin to the insect midgut.

MCSE-U-Net: multi-convolution blocks and squeeze and excitation blocks for vessel segmentation.

Background: Capturing the segmentation of blood vessels by a fundus camera is crucial for the medical evaluation of various retinal vascular issues. However, due to the complicated vascular structure and unclear clinical criteria, the precise segmentation of blood arteries remains very challenging. Methods: To address this issue, we developed the upgraded multi-convolution block and squeeze and excitation based on the U-shape network (MCSE-U-net) model that segments retinal vessels using a U-shaped network. This model uses multi-convolution (MC) blocks, squeeze and excitation (SE) blocks, and squeeze blocks. First, the input image was processed using the luminance, chrominance-blue, chrominance-red (YCbCr) color conversion method to further improve visibility. Second, a MC module was added to increase the model's ability to accurately segment blood vessels. Third, SE blocks were added to enhance the network model's ability to segment fine blood vessels in medical images. Results: The suggested architecture was assessed using evaluation metrics, including the Dice coefficient, sensitivity (sen), specificity (spe), accuracy (acc), and mean intersection over union (mIoU), on an open-source Digital Retinal Images for Vessel Extraction (DRIVE) data set. The outcomes showed the effectiveness of the suggested approach, particularly in the extraction of peripheral vascular anatomy. Using the suggested architecture, the model had a Dice coefficient of 0.8430, a sen of 0.8752, a spe of 0.9902, an acc of 0.9725, and a mIoU of 0.8473 for the DRIVE data set. The Dice coefficient, sen, spe, acc, and mIoU of the MCSE-U-net increased by 3.08%, 6.22%, 0.62%, 0.61%, and 3.01%, respectively, compared to the original U-net, demonstrating the better all-around performance of the MCSE-U-net. Conclusions: The MCSE-U-net network performed and achieved more than the technologies already in use.

Screening and Identification of Anti-Idiotypic Nanobody Capable of Broad-Spectrum Recognition of the Toxin Binding Region of Lepidopteran Cadherins and Mimicking Domain II of Cry2Aa Toxin.

The widespread use of Bacillus thuringiensis toxins as insecticides has brought about resistance problems. Anti-idiotypic nanobody approaches provide new strategies for resistance management and toxin evolution. In this study, the monoclonal antibody generated against the receptor binding region Domain II of Cry2Aa toxin was used as a target to screen materials with insecticidal activity. After four rounds of screening, anti-idiotypic nanobody 1C12 was obtained from the natural alpaca nanobody phage display library. To better analyze the activity of 1C12, soluble 1C12 was expressed by the Escherichia coli BL21 (DE3). The results showed that 1C12 not only binds the midgut brush border membrane vesicles (BBMV) of two lepidopteran species and cadherin CR9-CR11 of three lepidopteran species but also inhibits Cry2Aa toxins from binding to CR9-CR11. The insect bioassay showed that soluble 1C12 caused 25.65% and 23.61% larvae mortality of Helicoverpa armigera and Plutella xylostella, respectively. Although 1C12 has low insecticidal activity, soluble 1C12 possesses the ability to screen a broad-spectrum recognition of the toxin binding region of lepidopteran cadherins and can be used for the identification of the toxin binding region of other lepidopteran cadherins and the subsequent evolution of Cry2Aa toxin. The present study demonstrates a new strategy to screen for the production of novel insecticides.

Establishment of novel receptor-antibody sandwich assays to broadly detect Bacillus thuringiensis Cry1 and Cry2 toxins.

Bacillus thuringiensis (Bt) Cry toxins have been widely used in the development of genetically modified organisms (GMOs) for pest control. This work aimed to establish more cost effective and broader detection methods for commonly used Cry toxins. Using ligand blot and bio-layer interferometry, we confirmed that a recombinant toxin-binding fragments derived from Helicoverpa armigera cadherin-like protein (HaCad-TBR) could broadly bind Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab with the affinity of 0.149, 0.402, 120, and 4.12 nM, respectively. Based on the affinity results, a novel receptor-antibody sandwich assay broadly detecting Cry1A and Cry2 toxins was developed by using HaCad-TBR as capture molecules, and anti-Cry1A/Cry2A polyclonal antibodies (pAbs) as the detection antibodies. The detection limit (LOD) for Cry1Ab, Cry1Ab, Cry2Aa, and Cry2Ab were 5.30, 5.75, 30.83 and 13.70 ng/mL. To distinguish Cry1A and Cry2A toxins in a singular test, anti-Cry1A pAbs and anti-Cry2A pAbs were labelled with different quantum dots (QDs). The LOD for the four toxins by receptor-QDs-pAbs sandwich assay were calculated to be 1.36, 4.71, 17.48, and 7.54 ng/mL, respectively. The two developed methods were validated by spiked rice and corn samples, suggesting they may potentially be used in monitoring and quantifying Cry toxins in food and environment.

Generation of anti-idiotypic antibodies mimicking Cry2Aa toxin from an immunized mouse phage display library as potential insecticidal agents against Plutella xylostella.

This study tried to generate anti-idiotypic antibodies (Ab2s) which mimic Cry2Aa toxin using a phage-display antibody library (2.8 × 107 CFU/mL). The latter was constructed from a mouse immunized with F (ab')2 fragments digested from anti-Cry2Aa polyclonal antibodies. The F (ab')2 fragments and Plutella xylostella (P. xylostella) brush border membrane vesicles (BBMV) were utilized as targets for selection. Eight mouse phage-display single-chain variable fragments (scFvs) were isolated and identified by enzyme-linked immunoassay (ELISA), PCR and DNA sequencing after four rounds of biopanning. Among them, M3 exhibited the highest binding affinity with F (ab')2, while M4 bound the best with the toxin binding region of cadherin of P. xylostella (PxCad-TBR). Both of these two fragments were chosen for prokaryotic expression. The expressed M3 and M4 proteins with molecular weights of 30 kDa were purified. The M4 showed a binding affinity of 29.9 ± 2.4 nM with the PxCad-TBR and resulted in 27.8 ± 4.3 % larvae mortality against P. xylostella. Computer-assisted molecular modeling and docking analysis showed that mouse scFv M4 mimicked some Cry2Aa toxin binding sites when interacting with PxCad-TBR. Therefore, anti-idiotypic antibodies generated by BBMV-based screening could be useful for the development of new bio-insecticides as an alternative to Cry2Aa toxin for pest control.

Establishment of monoclonal antibody and scFv immuno-based assay for Cry2Aa toxin in spiked grain samples.

Bacillus thuringiensis (Bt) Cry toxins have been widely used in the development of genetically modified organisms (GMOs) for pest control. This work aimed to establish more cost effective methods for used Cry2Aa toxins. Three immunoassay methods (IC-ELISA, DAS-ELISA, and CLEIA) were successfully developed in this work. The mAb was used as the detecting antibody, for the IC-ELISA, the range of IC20 to IC80 was 1.11 µg/mL - 60.70 µg/mL, and an IC50 of 10.65 µg/mL. For the DAS-ELISA, the limit of detection (LOD) and limit of quantitation (LOQ) were 10.76 ng/mL and 20.70 ng/mL, respectively. For the CLEIA, the LOD and LOQ were 6.17 ng/mL and 7.40 ng/mL, respectively. The scFv-based detections were the most sensitive for detecting Cry2Aa. The LOD and LOQ for the DAS-ELISA were 118.75 ng/mL and 633.48 ng/mL, respectively. The LOD and LOQ for the CLEIA, read as 37.47 ng/mL and 70.23 ng/mL, respectively. The fact that Cry2Aa toxin was recovered in spiked grain samples further demonstrated that the approaches might be used to identify field samples. These methods provided good sensitivity, stability, and applicability for detecting Cry2Aa toxin, promising ultrasensitive monitoring and references for Cry toxins risk assessment.

Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) quantification of transient ischemia using a combination method of 5-pool Lorentzian fitting and inverse Z-spectrum analysis.

Background: Chemical exchange saturation transfer (CEST) is a promising method for the detection of biochemical alterations in cancers and neurological diseases. However, the sensitivity of the currently existing quantitative method for detecting ischemia needs further improvement. Methods: To further improve the quantification of the CEST signal and enhance the CEST detection for ischemia, we used a quantitative analysis method that combines an inverse Z-spectrum analysis and a 5-pool Lorentzian fitting. Specifically, a 5-pool Lorentzian simulation was conducted with the following brain tissue parameters: water, amide (3.5 ppm), amine (2.2 ppm), magnetization transfer (MT), and nuclear Overhauser enhancement (NOE; -3.5 ppm). The parameters were first calculated offline and stored as the initial value of the Z-spectrum fitting. Then, the measured Z-spectrum with the peak value set to 0 was fitted via the stored initial value, which yielded the reference Z-spectrum. Finally, the difference between the inverse of the Z-spectrum and the inverse of the reference Z-spectrum was used as the CEST definite spectrum. Results: The simulation results demonstrated that the Z-spectra of the rat brain were well simulated by a 5-pool Lorentzian fitting. Further, the proposed method detected a larger difference than did either the saturation transfer difference or the 5-pool Lorentzian fitting, as demonstrated by simulations. According to the results of the cerebral ischemia rat model, the proposed method provided the highest contrast-to-noise ratio (CNR) between the contralateral and the ipsilateral striatum under various acquisition conditions. The results indicated that the difference of fitted amplitudes generated with a 5-pool Lorentzian fitting in amide at 3.5 ppm (6.04%±0.39%; 6.86%±0.39%) was decreased in a stroke lesion compared to the contralateral normal tissue. Moreover, the difference of the residual of inversed Z-spectra in which 5-pool Lorentzian fitting was used to calculate the reference Z-spectra ( M T R R e x 5 L ) amplitudes in amide at 3.5 ppm (13.83%±2.20%, 15.69%±1.99%) was reduced in a stroke lesion compared to the contralateral normal tissue. Conclusions: M T R R e x 5 L is predominantly pH-sensitive and is suitable for detecting tissue acidosis following an acute stroke.

[Targeted innovative design of Bt Cry toxin insecticidal mimics].

Bt Cry toxin is the mostly studied and widely used biological insect resistance protein, which plays a leading role in the green control of agricultural pests worldwide. However, with the wide application of its preparations and transgenic insecticidal crops, the resistance to target pests and potential ecological risks induced by the drive are increasingly prominent and attracting much attention. The researchers seek to explore new insecticidal protein materials that can simulate the insecticidal function of Bt Cry toxin. This will help to escort the sustainable and healthy production of crops, and relieve the pressure of target pests' resistance to Bt Cry toxin to a certain extent. In recent years, the author's team has proposed that Ab2ß anti-idiotype antibody has the property of mimicking antigen structure and function based on the "Immune network theory" of antibody. With the help of phage display antibody library and specific antibody high-throughput screening and identification technology, Bt Cry toxin antibody was designed as the coating target antigen, and a series of Ab2ß anti-idiotype antibodies (namely Bt Cry toxin insecticidal mimics) were screened from the phage antibody library. Among them, the lethality of Bt Cry toxin insecticidal mimics with the strongest activity was close to 80% of the corresponding original Bt Cry toxin, showing great promise for the targeted design of Bt Cry toxin insecticidal mimics. This paper systematically summarized the theoretical basis, technical conditions, research status, and discussed the development trend of relevant technologies and how to promote the application of existing achievements, aiming to facilitate the research and development of green insect-resistant materials.

Improved sensitivity of the anti-microcystin-LR ELISA using phage-displayed alpha-type anti-idiotypic nanobody.

Anti-idiotypic antibodies (Ab2) are valuable tools that can be used for a better understanding of molecular mimicry and the immunological network. In this work, we showed a new application of a phage-displayed alpha-type Ab2 (Ab2α) to improve the sensitivity of an enzyme-linked immunosorbent assay (ELISA) detecting cyanobacterial toxin microcystin-LR (MC-LR). A monoclonal antibody (mAb) against MC-LR was used as an antigen to isolate binders in a camelid nanobody library. After three rounds of panning, three unique clones with strong binding against anti-MC-LR mAbs were isolated. These clones could specifically bind to anti-MC-LR mAbs without influencing mAbs binding with MC-LR, meaning these clones were Ab2αs. Based on the signal amplification effect of phage coat proteins and the non-competitive nature of Ab2α, a novel competitive ELISA method for MC-LR was established with a phage-displayed Ab2α. It showed that the phage-displayed Ab2α greatly enhanced the ELISA signal and sensitivity of the method was improved 3.5-fold to the conventional one. Combining with the optimization of pre-incubation time, the optimized ELISA decreased its limit of detection (LOD) from 4.5 ng/mL to 0.8 ng/mL (5.6-fold improvement). This new application of Ab2α may potentially be employed to improve the sensitivity of immunoassays for other environmental pollutants.

Generation of Human Domain Antibody Fragments as Potential Insecticidal Agents against Helicoverpa armigera by Cadherin-Based Screening.

New insecticidal genes and approaches for pest control are a hot research area. In the present study, we explored a novel strategy for the generation of insecticidal proteins. The midgut cadherin of Helicoverpa armigera (H. armigera) was used as a target to screen materials that have insecticidal activity. After three rounds of panning, the phage-displayed human domain antibody B1F6, which not only binds to the H. armigera cadherin CR9-CR11 but also significantly inhibits Cry1Ac toxins from binding to CR9-CR11, was obtained from a phage-displayed human domain antibody (DAb) library. To better analyze the relevant activity of B1F6, soluble B1F6 protein was expressed by Escherichia coli BL21 (DE3). The cytotoxicity assays demonstrated that soluble B1F6 induced Sf9 cell death when expressing H. armigera cadherin on the cell membrane. The insect bioassay results showed that soluble B1F6 protein (90 µg/cm2) caused 49.5 ± 3.3% H. armigera larvae mortality. The midgut histological results showed that soluble B1F6 caused damage to the midgut epithelium of H. armigera larvae. The present study explored a new strategy and provided a basic material for the generation of new insecticidal materials.

Screening and identification of vancomycin anti-idiotypic antibodies for against Staphylococcus aureus from a human phage display domain antibody library.

Staphylococcus aureus is a common food-borne pathogenic microorganism that poses a serious threat to food quality and safety, and can do harm to human health. In the past, researchers relied on antibiotics to control Staphylococcus aureus, though very effective, yet it was also worrying in the aspect of bio-safety. In fact, anti-idiotypic antibody (Anti-Id) shows its potential to mimic some of the structural and biological functions of antigens. Therefore, in this study, based on Anti-Id theory and technology, we expect to obtain the vancomycin Anti-Id which can mimic vancomycin against Staphylococcus aureus from a human phage display domain antibody library. After four rounds of bio-panning, a total of 18 positive Anti-Ids were obtained. Among them, two Anti-Ids named Anti-Id-2C12 and Anti-Id-1F5 were identified as "ß" type Anti-Ids, and afterwards they were selected for gene cloning and protein expression in prokaryotic expression system. As a result, a concentration of purified proteins with 568.6 µg/mL (Anti-Id-2C12) and 602.3 µg/mL (Anti-Id-1F5) were successfully obtained, and their minimum inhibitory concentration (MIC) values for Staphylococcus aureus were 125 and 200 µg/mL, respectively. As they are human heavy-chain domain antibodies, which were theoretically harmless to humans, they have the potential application value as preservatives in food and edible agricultural products.

Identification of single domain antibodies with insect cytotoxicity using phage-display antibody library screening and Plutella xylostella ATP-binding cassette transporter subfamily C member 2 (ABCC2) -based insect cell expression system.

It is extremely imminent to study a new strategy to manage agricultural pest like Plutella xylostella (P. xylostella) which is currently resistant to most of pesticides, including three domain-Cry toxins from Bacillus thuringiensis (Bt). In this study, we reported a phage displayed single domain antibody screening from human domain antibody (DAb) library targeted on Spodoptera frugiperda 9 (Sf9) cells expressed Cry1Ac toxin receptor, ATP-dependent binding cassette transporter C2 in P. xylostella (PxABCC2). After three rounds of panning, three cytotoxic antibodies (1D2, 2B7, 3C4) were obtained from thirty-eight antibodies and displayed high binding ability towards PxABCC2-expressed Sf9 cells. Through homology modeling and molecular docking, the interaction mode indicated that the most cytotoxic 1D2 of the three antibodies presented the lowest binding free energy required and had the most hydrogen bond formed with PxABCC2 in molecular docking analysis. Functional assay of key regions in 1D2 via Alanine replacement indicated that complementarity-determining region (CDR) 3 played a crucial role in antibody exerts binding activity and cytotoxicity. This study provides the first trial for discovering of potential cytotoxic antibodies from the human antibody library via specific receptor-expressed insect cell system biopanning.

Epitopes prediction for microcystin-LR by molecular docking.

Microcystin-LR (MC-LR) is one of the most worldwide harmful cyanobacterial toxins. A lots of antibodies against MC-LR have been generated and characterized. However, the knowledge about the epitopes of MC-LR was still limited. The objective of this study was to analyze the epitopes of MC-LR and demonstrate the binding mode of MC-LR with its antibody. The variable genes of a mouse hybridoma cell line (Mab5H1-3B3) raised against MC-LR have been cloned and assembled in a single chain variable fragment (scFv), and then soluble expressed in E.coli BL21. Based on the scFv, the IC50 and IC10 for MC-LR were determined to be 7.45 nM and 0.30 nM by competitive ELISA. And the scFv also showed 115% and 112% cross-reactivities to MC-RR and MC-YR, and 59% to MC-LA. By molecular docking, the binding mode between MC-LR and its scFv was demonstrated. A hydrogen bond interaction was observed between the carbonyl group of Adda5 residue of MC-LR and its scFv, and the guanidyl group of Arg4 residue and phenyl group of Adda5 residue of MC-LR were also involved in the interaction. These predicted epitopes were supported by antibody cross-reactivity data. By comparing the antibody informatics of MC-LR scFv with its predicted paratopes, VH-CDR1 was crucial for MC-LR binding, and its specificity could be tuned by engineering in Vκ-CDR1 and Vκ-CDR3. These information would be useful for the hapten design for microcystins or improving the properties of MC-LR scFv in vitro.

Docking-based generation of antibodies mimicking Cry1A/1B protein binding sites as potential insecticidal agents against diamondback moth (Plutella xylostella).

BACKGROUND: Broad use of insecticidal Cry proteins from Bacillus thuringiensis in biopesticides and transgenic crops has resulted in cases of practical field resistance, highlighting the need for novel approaches to insect control. Previously we described an anti-Cry1Ab idiotypic-antibody (B12-scFv) displaying toxicity against rice leafroller (Cnaphalocrocis medinalis) larvae, supporting the potential of antibodies for pest control. The goal of the present study was to generate insecticidal antibodies against diamondback moth (Plutella xylostella) larvae. RESULTS: Four genetically engineered antibodies (GEAbs) were designed in silico from B12-scFv using three-dimensional (3D) structure and docking predictions to alkaline phosphatase (ALP) as a Cry1Ac receptor in P. xylostella. Among these GEAbs, the GEAb-dVL antibody consisting of two light chains had overlapping binding sites with Cry1A and Cry1B proteins and displayed high binding affinity to P. xylostella midgut brush border membrane (BBM) proteins. Proteins in BBM identified by pull-down assays as binding to GEAb-dVL included an ABC transporter and V-ATPase subunit A protein. Despite lacking the α-helical structures in Cry1A that are responsible for pore formation, ingestion of GEAb-dVL disrupted the P. xylostella larval midgut epithelium and resulted in toxicity. Apoptotic genes were activated in gut cells upon treatment with GEAb-dVL . CONCLUSION: This study describes the first insecticidal GEAb targeting P. xylostella by mimicking Cry proteins. Data support that GEAb-dVL toxicity is associated to activation of intracellular cell death pathways, in contrast to pore-formation associated toxicity of Cry proteins. This work provides a foundation for the design of novel insecticidal antibodies for insect control. © 2021 Society of Chemical Industry.

Identification and characterization of alkaline phosphatase gene phoX in Microcystis aeruginosa PCC7806.

PhoX is an extracellular alkaline phosphatase that is widely found in cyanobacteria and plays an important role in the conversion of extracellular organophosphorus into soluble inorganic phosphorus. However, the phoX gene has not yet been experimentally confirmed to exist in bloom-forming Microcystis species. In this study, we identified a putative phoX gene (GenBank accession no. ARI79942.1) in M. aeruginosa PCC7806 and overexpressed it in Escherichia coli 21 (DE3). The expressed PhoX protein displayed phosphodiesterase and phosphomonoesterase activities. In contrast to other bacterial PhoX proteins, which are activated mainly by Ca2+, Microcysits PhoX was most strongly activated by Mg2+, followed by Co2+, Ca2+, Zn2+ and Mn2+, but it was inhibited by Ni2+. Sequence analysis showed that phoX was highly conserved in the Microcystis genus (DNA similarity > 96% between species). phoX expression responded significantly to different environmental phosphorus levels. When PCC7806 cells were cultured in phosphorus-deficient medium (BG11-P), phoX expression reached its highest level at 2 h and then decreased to a low level at 4 h. Organophosphate induced the expression of phoX; its expression reached the highest level at 4 h and was maintained at a high level at 6 h. Our results confirmed a putative phoX gene and demonstrated that the phoX gene of Microcystis is conserved.

Anti-idiotypic single-chain variable fragment antibody partially mimic the functionally spatial structure of Cry2Aa toxin.

The anti-idiotypic antibody is widely used in the field of immunology to simulate structural features or even induce the biological activity of antigens. In this study, we obtained seven anti-idiotypic single-chain variable fragments (scFv) antibodies of Cry2Aa toxin from a phage-displayed mutant library constructed using error-prone PCR technique. A mutant designated 2-12B showed the best binding ability amongst all anti-idiotypic scFv isolates to Plutella xylostella brush border membrane vesicles (BBMVs). 2-12B and Cry2Aa toxin shared a potential receptor of polycalin in P. xylostella BBMVs. Homology modeling and molecular docking demonstrated that 2-12B and Cry2Aa toxin have seven common binding amino acid residues in polycalin. Insect bioassay results suggested that 2-12 had insecticidal efficacy against P. xylostella larvae. These results indicated that the Cry2Aa anti-idiotypic scFv antibody 2-12B partially mimicked the structure and function of Cry2Aa toxin. The anti-idiotypic scFv antibody provides the basic material for the future study of surrogate molecules or new insecticidal materials.

Microcystin-LR heterologous genetically engineered antibody recombinant and its binding activity improvement and application in immunoassay.

Microcystin-LR (MC-LR) is a high-toxic biohazard that pollutes ecological environment and agroproducts. In this study, a newly recombined genetically engineered antibody (AVHH-MVH) with higher thermal stability and binding activity was designed by chain shuffling and based on our previously obtained anti-MC-LR scFv and nanobody. Based on AVHH-MVH template, a capacity of 8.99 × 105 CFU/mL of phage display AVHH-MVH mutagenesis library was constructed by site-directed mutagenesis in MVH-CDR3 region, and then used for ultrasensitive mutants screening. Afterwards, a total of five positive AVHH-MVH mutants were isolated from the mutagenesis library, and their binding activity was higher than AVHH-MVH for MC-LR. The AVHH-MVH mutant 3 was cloned into pET-25b vector for soluble expression, and the concentration of target protein expressed in culture system was 43.5 mg/L. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was established based on purified AVHH-MVH mutant 3 protein, and it showed ultrasensitive binding activity for MC-LR with the detection limit of 0.0075 µg/L, which was far below the maximum residue limit standard of 1.0 µg/L in drinking water proposed by World Health Organization. The established IC-ELISA shows good accuracy, repeatability, stability and applicability for MC-LR spiked samples, and it is promising for MC-LR ultrasensitive monitoring.

Construction and characterization of a class-specific single-chain variable fragment against pyrethroid metabolites.

Pyrethroids are insecticides that are widely used in rural and urban areas worldwide. After entering the environment, pyrethroids are rapidly metabolized or degraded by various biological or abiotic methods. In this study, a single-chain variable fragment (scFv) which could simultaneously detect three pyrethroid metabolites was constructed based on a hybridoma raised against 3-phenoxybenzoic acid (3-PBA). By molecular docking, it showed that there were hydrogen bonds, hydrophobic interactions, CH-π interaction, and cation-π interaction between 3-PBA and its scFv. All the contact residues contributing to hydrogen bonds are located in VH-CDR2 or its neighboring region, and two of them were mutants of the closest germline sequence. Based on competitive ELISA, the half maximal inhibitory concentration (IC50) of the scFv for 3-PBA, 3-phenoxybenzaldehyde (PBAld), and 3-phenoxybenzyl alcohol (PBAlc) were calculated to be 0.55, 0.59, and 0.63 µgmL-1, respectively. The scFv also showed 23.91%, 13.41%, 1.15%, 1.00%, and 0.56% cross-reactivity with phenothrin, deltamethrin, fenvalerate, beta-cypermethrin, and fenpropathrin. The broad specificity of the scFv may be due to its hapten design. The scFv could be employed in class-specific immunoassays for pyrethroid metabolites with phenoxybenzyl (PB) group. It is also potentially used for characterizing degradation of pyrethroids or detecting PBAlc (PBAld) alone, and the detection results should be confirmed by other selective methods. KEY POINTS: • A scFv which can simultaneously detect 3-PBA, PBAlc, and PBAld was constructed. • Antibody informatics and binding mode of the scFv were obtained. • The reason for its broad specificity was discussed. • It could be used to monitor single or multi-pyrethroid metabolites with PB group.

Cell wall surface layer (S-layer) promotes colony formation in Microcystis: comparison of S-layer characteristics between colonial and unicellular forms of Microcystis and function conformation.

Colony is a key to Microcystis becoming a dominant population and forming blooms. To find the mechanism of colony formation, we investigated cell wall structures of colonial and unicellular strains. Results showed that colonial strains had significant surface layer protein (S-layer) on the surface of cells than unicellular strains by transmission electron microscopy. Western blot showed colonial strains had more S-layer than the unicellular strains. When the S-layer gene (GenBank accession number CAO89090.1) of Microcystis aeruginosa PCC7806 was expressed in Synechocystis sp. PCC6803, PCC6803 aggregated into colonial morphology. The results indicated that the S-layer could promote colony formation in Microcystis. Based on the S-layer sequences of PCC6803 and PCC7806, nine S-layer genes in other Microcystis strains were screened from the GenBank. Sequence comparing showed that the S-layers conserved regions were all located in N-terminal. The S-layers contain repeats-in-toxin (RTX) sequences with Ca2+-binding site, and their amino acid composition, hydrophobicity, isoelectric point, etc. were consistent with the characteristics of RTX-type S-layer in bacteria.