Natural photoreceptors as a source of fluorescent proteins, biosensors, and optogenetic tools.

Genetically encoded optical tools have revolutionized modern biology by allowing detection and control of biological processes with exceptional spatiotemporal precision and sensitivity. Natural photoreceptors provide researchers with a vast source of molecular templates for engineering of fluorescent proteins, biosensors, and optogenetic tools. Here, we give a brief overview of natural photoreceptors and their mechanisms of action. We then discuss fluorescent proteins and biosensors developed from light-oxygen-voltage-sensing (LOV) domains and phytochromes, as well as their properties and applications. These fluorescent tools possess unique characteristics not achievable with green fluorescent protein-like probes, including near-infrared fluorescence, independence of oxygen, small size, and photosensitizer activity. We next provide an overview of available optogenetic tools of various origins, such as LOV and BLUF (blue-light-utilizing flavin adenine dinucleotide) domains, cryptochromes, and phytochromes, enabling control of versatile cellular processes. We analyze the principles of their function and practical requirements for use. We focus mainly on optical tools with demonstrated use beyond bacteria, with a specific emphasis on their applications in mammalian cells.

Sequence grammar underlying the unfolding and phase separation of globular proteins.

Aberrant phase separation of globular proteins is associated with many diseases. Here, we use a model protein system to understand how the unfolded states of globular proteins drive phase separation and the formation of unfolded protein deposits (UPODs). We find that for UPODs to form, the concentrations of unfolded molecules must be above a threshold value. Additionally, unfolded molecules must possess appropriate sequence grammars to drive phase separation. While UPODs recruit molecular chaperones, their compositional profiles are also influenced by synergistic physicochemical interactions governed by the sequence grammars of unfolded proteins and cellular proteins. Overall, the driving forces for phase separation and the compositional profiles of UPODs are governed by the sequence grammars of unfolded proteins. Our studies highlight the need for uncovering the sequence grammars of unfolded proteins that drive UPOD formation and cause gain-of-function interactions whereby proteins are aberrantly recruited into UPODs.

A novel method for measurements of sleep/wake states, feeding and drinking behaviors using the tracking technique of 3D positions in freely moving mice.

We have previously developed a 3D video tracking system which enables us to analyze long-term quantitative analysis of gene expression in freely moving mice. In the present study, we improved 3D video tracking and developed a system that analyzes more detailed behavioral data. We succeeded in simultaneously analyzing sleep-wake, feeding, and drinking behavior rhythms in the same individual using our tracking system. This system will make it possible to measure gene expression in each tissue in vivo in real time in relation to the various behavioral rhythms mentioned above.

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.

OptoProfilin: A Single Component Biosensor of Applied Cellular Stress.

The actin cytoskeleton is a biosensor of cellular stress and a potential prognosticator of human disease. In particular, aberrant cytoskeletal structures such as stress granules formed in response to energetic and oxidative stress are closely linked to ageing, cancer, cardiovascular disease, and viral infection. Whether these cytoskeletal phenomena can be harnessed for the development of biosensors for cytoskeletal dysfunction and, by extension, disease progression, remains an open question. In this work, we describe the design and development of an optogenetic iteration of profilin, an actin monomer binding protein with critical functions in cytoskeletal dynamics. We demonstrate that this optically activated profilin ('OptoProfilin') can act as an optically triggered biosensor of applied cellular stress in select immortalized cell lines. Notably, OptoProfilin is a single component biosensor, likely increasing its utility for experimentalists. While a large body of preexisting work closely links profilin activity with cellular stress and neurodegenerative disease, this, to our knowledge, is the first example of profilin as an optogenetic biosensor of stress-induced changes in the cytoskeleton.

CRY2 and FBXL3 Cooperatively Degrade c-MYC.

For many years, a connection between circadian clocks and cancer has been postulated. Here we describe an unexpected function for the circadian repressor CRY2 as a component of an FBXL3-containing E3 ligase that recruits T58-phosphorylated c-MYC for ubiquitylation. c-MYC is a critical regulator of cell proliferation; T58 is central in a phosphodegron long recognized as a hotspot for mutation in cancer. This site is also targeted by FBXW7, although the full machinery responsible for its turnover has remained obscure. CRY1 cannot substitute for CRY2 in promoting c-MYC degradation. Their unique functions may explain prior conflicting reports that have fueled uncertainty about the relationship between clocks and cancer. We demonstrate that c-MYC is a target of CRY2-dependent protein turnover, suggesting a molecular mechanism for circadian control of cell growth and a new paradigm for circadian protein degradation.

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.

V-ATPase E mediates Cry2Ab binding and toxicity in Helicoverpa armigera.

Cry2Ab is one of the important alternative Bt proteins that can be used to manage insect pests resistant to Cry1A toxins and to expand the insecticidal spectrum of pyramided Bt crops. Previous studies have showed that vacuolar H+-ATPase subunits A and B (V-ATPase A and B) may be involved in Bt insecticidal activities. The present study investigated the role of V-ATPases subunit E in the toxicity of Cry2Ab in Helicoverpa amigera. RT-PCR analysis revealed that oral exposure of H. amigera larvae to Cry2Ab led to a significant reduction in the expression of H. armigera V-ATPase E (HaV-ATPase E). Ligand blot, homologous and heterologous competition experiments confirmed that HaV-ATPases E physically and specifically bound to activated Cry2Ab toxin. Heterologous expressing of HaV-ATPase E in Sf9 cells made the cell line more susceptible to Cry2Ab, whereas knockdown of the endogenous V-ATPase E in H. zea midgut cells decreased Cry2Ab's cytotoxicity against this cell line. Further in vivo bioassay showed that H. armigera larvae fed a diet overlaid with both Cry2Ab and E. coli-expressed HaV-ATPase E protein suffered significantly higher mortality than those fed Cry2Ab alone. These results support that V-ATPases E is a putative receptor of Cry2Ab and can be used to improve Cry2Ab toxicity and manage Cry2Ab resistance at least in H. armigera.

Cadherin Is a Binding Protein but Not a Functional Receptor of Bacillus thuringiensis Cry2Ab in Helicoverpa armigera.

Midgut receptors play a critical role in the specificity of Cry toxins for individual insect species. Cadherin proteins are essential putative receptors of Cry1A toxins in lepidopteran larvae. Cry2A family members share common binding sites in Helicoverpa armigera, and one of them, Cry2Aa, has been widely reported to interact with midgut cadherin. Here, we studied the binding interaction and functional role of H. armigera cadherin in the mechanism of Cry2Ab toxicity. A region spanning from cadherin repeat 6 (CR6) to the membrane-proximal region (MPR) of cadherin protein was produced as six overlapping peptides to identify the specific binding regions of Cry2Ab. Binding assays showed that Cry2Ab binds nonspecifically to peptides containing CR7 and CR11 regions in a denatured state but binds specifically only to CR7-containing peptides in the native state. The peptides CR6-11 and CR6-8 were transiently expressed in Sf9 cells to assess the functional role of cadherin. Cytotoxicity assays showed that Cry2Ab is not toxic to the cells expressing any of the cadherin peptides. However, ABCA2-expressing cells showed high sensitivity to Cry2Ab toxin. Neither increased nor decreased sensitivity to Cry2Ab was observed when the peptide CR6-11 was coexpressed with the ABCA2 gene in Sf9 cells. Instead, treating ABCA2-expressing cells with a mixture of Cry2Ab and CR6-8 peptides resulted in significantly reduced cell death compared with treatment with Cry2Ab alone. Moreover, silencing of the cadherin gene in H. armigera larvae showed no significant effect on Cry2Ab toxicity, in contrast to the reduced mortality in ABCA2-silenced larvae. IMPORTANCE To improve the efficiency of production of a single toxin in crops and to delay the evolution of insect resistance to the toxin, the second generation of Bt cotton, expressing Cry1Ac and Cry2Ab, was introduced. Understanding the mode action of the Cry proteins in the insect midgut and the mechanisms insects use to overcome these toxins plays a crucial role in developing measures to counter them. Extensive studies have been conducted on the receptors of Cry1A toxins, but relatively little has been done about those of Cry2Ab. By showing the nonfunctional binding of cadherin protein with Cry2Ab, we have furthered the understanding of Cry2Ab receptors.

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.

The dual-action mechanism of Arabidopsis cryptochromes.

Photoreceptor cryptochromes (CRYs) mediate blue-light regulation of plant growth and development. It has been reported that Arabidopsis CRY1and CRY2 function by physically interacting with at least 84 proteins, including transcription factors or co-factors, chromatin regulators, splicing factors, messenger RNA methyltransferases, DNA repair proteins, E3 ubiquitin ligases, protein kinases and so on. Of these 84 proteins, 47 have been reported to exhibit altered binding affinity to CRYs in response to blue light, and 41 have been shown to exhibit condensation to CRY photobodies. The blue light-regulated composition or condensation of CRY complexes results in changes of gene expression and developmental programs. In this mini-review, we analyzed recent studies of the photoregulatory mechanisms of Arabidopsis CRY complexes and proposed the dual mechanisms of action, including the "Lock-and-Key" and the "Liquid-Liquid Phase Separation (LLPS)" mechanisms. The dual CRY action mechanisms explain, at least partially, the structural diversity of CRY-interacting proteins and the functional diversity of the CRY photoreceptors.

Optogenetically-induced multimerization of the dopamine transporter increases uptake and trafficking to the plasma membrane.

The dopamine transporter (DAT) is essential for the reuptake of the released neurotransmitter dopamine (DA) in the brain. Psychostimulants, methamphetamine and cocaine, have been reported to induce the formation of DAT multimeric complexes in vivo and in vitro. The interpretation of DAT multimer function has been primarily in the context of compounds that induce structural and functional modifications of the DAT, complicating the understanding of the significance of DAT multimers. To examine multimerization in the absence of DAT ligands as well as in their presence, we developed a novel, optogenetic fusion chimera of cryptochrome 2 and DAT with an mCherry fluorescent reporter (Cry2-DAT). Using blue light to induce Cry2-DAT multimeric protein complex formation, we were able to simultaneously test the functional contributions of DAT multimerization in the absence or presence of substrates or inhibitors with high spatiotemporal precision. We found that blue light-stimulated Cry2-DAT multimers significantly increased IDT307 uptake and MFZ 9-18 binding in the absence of ligands as well as after methamphetamine and nomifensine treatment. Blue light-induced Cry2-DAT multimerization increased colocalization with recycling endosomal marker Rab11 and had decreased presence in Rab5-positive early endosomes and Rab7-positive late endosomes. Our data suggest that the increased uptake and binding results from induced and rapid trafficking of DAT multimers to the plasma membrane. Our data suggest that DAT multimers may function to help maintain DA homeostasis.

Molecular cloning of a new cry2A-type gene from Bacillus thuringiensis strain Nn10 and its expression studies.

In the present study, eight indigenous Bacillus thuringiensis isolates of Western Ghats of India with more than 90% toxicity against Helicoverpa armigera were characterized for cry2A gene sub families. Seven of the eight isolates harboured cry2Aa, cry2Ab and cry2Ac genes alone and or in combination. Further, the indigenous cry2Aa gene(s) from Bacillus thuringiensis isolate Nn10 which showed 100% mortality against Helicoverpa armigera was cloned and expressed into recombinant Bt strains for management of resistance development in insects. The ORF of cry2Aa (∼1.9 kb) gene(s) from Nn10 isolate was ligated with T/A vector (pTZ57 R/T) and expressed in E. coli, DH5α. Automated sequence analysis of newly cloned recombinant cry2Aa revealed 99% homology to 916 bases in the 3' region of minus strand and 100% homology with 720 bases in the 5' region of holotype cry2Aa1 gene. The partial Cry2Aa amino acid sequence of Bt strain, Nn10, deduced from the nucleotide sequence generated by M13F primer showed four amino acid variation in comparison to Cry2Aa1 holotype, at 338, 345, 346 and 489th position of ORF and the sequence was submitted to the NCBI. Further the expression of ORF of cry2Aa of Nn10 into acrystalliferous Bt strain, 4Q7 using expression vector pHT3P2T under the transcriptional control of cry3Aa promoter and cry2Aa terminator. SDS PAGE analysis of recombinant protein exhibited a prominent band of about 65 kDa. Bioassay studies revealed that recombinant proteins, Cry2Aa of Nn10 was toxic to Helicoverpa armigera with LC50 value of 7.26 µg ml-1.

Optogenetic Control of PIP2 Interactions Shaping ENaC Activity.

The activity of the epithelial Na+ Channel (ENaC) is strongly dependent on the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 binds two distinct cationic clusters within the N termini of ß- and γ-ENaC subunits (ßN1 and γN2). The affinities of these sites were previously determined using short synthetic peptides, yet their role in sensitizing ENaC to changes in PIP2 levels in the cellular system is not well established. We addressed this question by comparing the effects of PIP2 depletion and recovery on ENaC channel activity and intracellular Na+ levels [Na+]i. We tested effects on ENaC activity with mutations to the PIP2 binding sites using the optogenetic system CIBN/CRY2-OCRL to selectively deplete PIP2. We monitored changes of [Na+]i by measuring the fluorescent Na+ indicator, CoroNa Green AM, and changes in channel activity by performing patch clamp electrophysiology. Whole cell patch clamp measurements showed a complete lack of response to PIP2 depletion and recovery in ENaC with mutations to ßN1 or γN2 or both sites, compared to wild type ENaC. Whereas mutant ßN1 also had no change in CoroNa Green fluorescence in response to PIP2 depletion, γN2 did have reduced [Na+]i, which was explained by having shorter CoroNa Green uptake and half-life. These results suggest that CoroNa Green measurements should be interpreted with caution. Importantly, the electrophysiology results show that the ßN1 and γN2 sites on ENaC are each necessary to permit maximal ENaC activity in the presence of PIP2.

Impact of Caterpillar Increased Feeding Rates on Reduction of Bt Susceptibility.

The use of insect-resistant transgenic crops producing Bacillus thuringiensis protein Cry toxins (Bt) to control caterpillars is wide-spread. Development of a mechanism to prevent Bt from reaching its target site in the digestive system could result in Bt resistance and resistance to other insecticides active per os. Increased feeding rates by increasing temperature in tobacco budworms, Chloridea virescens, and bollworms, Helicoverpa zea, decreased Bt Cry1Ac susceptibility and mortality. The same was found in C. virescens for Bollgard II plant extract containing Bt Cry1Ac and Cry2Ab2 toxins. Furthermore, H. zea from the same inbred laboratory colony that fed faster independent of temperature manipulation were less susceptible to Bt intoxication. A laboratory derived C. virescens Bt resistant strain demonstrated a higher feeding rate on non-Bt artificial diet than the parental, Bt susceptible strain. A laboratory-reared Bt resistant fall armyworm, Spodoptera frugiperda, strain also fed faster on non-Bt diet compared to Bt susceptible caterpillars of the same species, both originally collected from corn. The studies in toto and the literature reviewed support the hypothesis that increased feeding rate is a behavioral mechanism for reducing caterpillar susceptibility to Bt. Its possible role in resistance needs further study.

CofActor: A light- and stress-gated optogenetic clustering tool to study disease-associated cytoskeletal dynamics in living cells.

The hallmarks of neurodegenerative diseases, including neural fibrils, reactive oxygen species, and cofilin-actin rods, present numerous challenges in the development of in vivo diagnostic tools. Biomarkers such as ß-amyloid (Aß) fibrils and Tau tangles in Alzheimer's disease are accessible only via invasive cerebrospinal fluid assays, and reactive oxygen species can be fleeting and challenging to monitor in vivo Although remaining a challenge for in vivo detection, the protein-protein interactions underlying these disease-specific biomarkers present opportunities for the engineering of in vitro pathology-sensitive biosensors. These tools can be useful for investigating early stage events in neurodegenerative diseases in both cellular and animal models and may lead to clinically useful reagents. Here, we report a light- and cellular stress-gated protein switch based on cofilin-actin rod formation, occurring in stressed neurons in the Alzheimer's disease brain and following ischemia. By coupling the stress-sensitive cofilin-actin interaction with the light-responsive Cry2-CIB blue-light switch, referred to hereafter as the CofActor, we accomplished both light- and energetic/oxidative stress-gated control of this interaction. Site-directed mutagenesis of both cofilin and actin revealed residues critical for sustaining or abrogating the light- and stress-gated response. Of note, the switch response varied depending on whether cellular stress was generated via glycolytic inhibition or by both glycolytic inhibition and azide-induced ATP depletion. We also demonstrate light- and cellular stress-gated switch function in cultured hippocampal neurons. CofActor holds promise for the tracking of early stage events in neurodegeneration and for investigating actin's interactions with other proteins during cellular stress.

MALAT1 recruited the E3 ubiquitin ligase FBXW7 to induce CRY2 ubiquitin-mediated degradation and participated in trophoblast migration and invasion.

This study aimed to investigate the mechanism by which MALAT1 regulates CRY2 expression and participates in trophoblast migration and invasion. Three patients with unexplained recurrent spontaneous abortion, four patients with missed abortion, and four women who underwent artificial miscarriages were enrolled in this study. Quantitative reverse-transcription polymerase chain reaction and western blot analysis were used to detect RNA and protein expression, respectively. Trophoblast migration and invasion were detected by wound-healing and transwell invasion assays. RNA pull-down and Co-IP assays were used to indicate the interaction between MALAT1 and FBXW7 or the interaction between FBXW7 and CRY2. The results showed significantly decreased MALAT1 expression in the villous specimens from the RSA patients relative to that in the villous specimens from the missed abortion patients and the normal villous specimens. MALAT1 promoted trophoblast cell migration and invasion by negatively regulating CRY2 protein expression. MALAT1 recruited FBXW7 to impair CRY2 protein stability. In conclusion, MALAT1 downregulation in trophoblasts might be related to miscarriage. MALAT1 may recruit the E3 ubiquitin ligase FBXW7 to induce CRY2 ubiquitin-mediated degradation and participate in trophoblast migration and invasion.

Low CLOCK and CRY2 in 2nd trimester human maternal blood and risk of preterm birth: a nested case-control study†.

Previous studies have observed an association between maternal circadian rhythm disruption and preterm birth (PTB). However, the underlying molecular mechanisms and the potential of circadian clock genes to serve as predictors of PTB remain unexplored. We examined the association of 10 core circadian transcripts in maternal blood with spontaneous PTB (sPTB) vs term births using a nested case-control study design. We used a public gene expression dataset (GSE59491), which was nested within the All Our Babies (AOB) study cohort in Canada. Maternal blood was sampled in Trimesters 2-3 from women with sPTB (n = 51) and term births (n = 106), matched for five demographic variables. In 2nd trimester maternal blood, only CLOCK and CRY2 transcripts were significantly lower in sPTB vs term (P = 0.02-0.03, false discovery rate (FDR) < 0.20). A change of PER3 mRNA from trimesters 2-3 was significantly associated with sPTB (decline in sPTB, P = 0.02, FDR < 0.20). When CLOCK and CRY2 were modeled together in 2nd trimester blood, the odds of being in the low level of both circadian gene transcripts was greater in sPTB vs term (OR = 4.86, 95%CI = (1.75,13.51), P < 0.01). Using GSVA and Pearson correlation, we identified 98 common pathways that were negatively or positively correlated with CLOCK and CRY2 expression (all P < 0.05, FDR < 0.10). The top three identified pathways were amyotrophic lateral sclerosis, degradation of extracellular matrix, and inwardly rectifying potassium channels. These three processes have previously been shown to be involved in neuron death, parturition, and uterine excitability during pregnancy, respectively.

Directed evolution approaches for optogenetic tool development.

Photoswitchable proteins enable specific molecular events occurring in complex biological settings to be probed in a rapid and reversible fashion. Recent progress in the development of photoswitchable proteins as components of optogenetic tools has been greatly facilitated by directed evolution approaches in vitro, in bacteria, or in yeast. We review these developments and suggest future directions for this rapidly advancing field.

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.