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 Ab2β 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 Ab2β 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.
Insecticides/metabolism* ; Bacillus thuringiensis ; Endotoxins/pharmacology* ; Bacillus thuringiensis Toxins/metabolism* ; Hemolysin Proteins/pharmacology* ; Bacterial Proteins/chemistry* ; Plants, Genetically Modified/genetics* ; Pest Control, Biological

Bacillus thuringiensis is widely used as an insecticide which is safe and environmentally friendly to humans and animals. One of the important insecticidal mechanisms is the binding of Bt toxins to specific toxin receptors in insect midgut and forming a toxin perforation which eventually leads to insect death. The resistance of target pests to Bt toxins is an important factor hampering the long-term effective cultivation of Bt crops and the continuous use of Bt toxins. This review summarizes the mechanism of insect resistance to Bt toxins from the perspective of important Bt toxin receptors in midgut cells of Lepidopteran insects, which may facilitate the in-depth study of Bt resistance mechanism and pest control.
Animals ; Bacillus thuringiensis/genetics* ; Bacillus thuringiensis Toxins ; Bacterial Proteins/metabolism* ; Endotoxins/metabolism* ; Hemolysin Proteins/metabolism* ; Insecta/metabolism* ; Insecticide Resistance/genetics* ; Insecticides/pharmacology* ; Pest Control, Biological

A transgenic maize event ZD12-6 expressing a Bacillus thuringiensis (Bt) fusion protein Cry1Ab/Cry2Aj and a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein G10 was characterized and evaluated. Southern blot analysis indicated that ZD12-6 is a single copy integration event. The insert site was determined to be at chromosome 1 by border sequence analysis. Expression analyses of Bt fusion protein Cry1Ab/Cry2Aj and the EPSPS protein G10 suggested that they are both expressed stably in different generations. Insect bioassays demonstrated that the transgenic plants are highly resistant to Asian corn borer (Ostrinia furnacalis), cotton boll worm (Helicoverpa armigera), and armyworm (Mythimna separata). This study suggested that ZD12-6 has the potential to be developed into a commercial transgenic line.
3-Phosphoshikimate 1-Carboxyvinyltransferase/metabolism* ; Animals ; Bacillus thuringiensis Toxins ; Bacterial Proteins/metabolism* ; China ; Disease Resistance/genetics* ; Drug Resistance/genetics* ; Endotoxins/metabolism* ; Gene Expression Profiling ; Glycine/chemistry* ; Hemolysin Proteins/metabolism* ; Insecta ; Plant Diseases/prevention & control* ; Plants, Genetically Modified/genetics* ; Zea mays/genetics* ; Glyphosate

OBJECTIVETo investigate the flexibility and mobility of the Bacillus thuringiensis toxin Cry1Aa.

METHODSThe graph theory-based program Constraint Network Analysis and normal mode-based program NMsim were used to analyze the global and local flexibility indices as well as the fluctuation of individual residues in detail.

RESULTSThe decrease in Cry1Aa network rigidity with the increase of temperature was evident. Two phase transition points in which the Cry1Aa structure lost rigidity during the thermal simulation were identified. Two rigid clusters were found in domains I and II. Weak spots were found in C-terminal domain III. Several flexible regions were found in all three domains; the largest residue fluctuation was present in the apical loop2 of domain II.

CONCLUSIONAlthough several flexible regions could be found in all the three domains, the most flexible regions were in the apical loops of domain II.

Bacillus thuringiensis ; Bacterial Proteins ; chemistry ; genetics ; metabolism ; Cluster Analysis ; Computer Simulation ; Endotoxins ; chemistry ; genetics ; metabolism ; Entropy ; Hemolysin Proteins ; chemistry ; genetics ; metabolism ; Models, Structural ; Mutation ; Protein Conformation ; Protein Unfolding ; Software ; Temperature

Exotoxins produced by Actinobacillus (A.) pleuropneumoniae (Apx) play major roles in the pathogenesis of pleuropneumonia in swine. This study investigated the role of ApxI in hemolysis and cellular damage using a novel apxIA mutant, ApxIA336, which was developed from the parental strain A. pleuropneumoniae serotype 10 that produces only ApxI in vitro. The genotype of ApxIA336 was confirmed by PCR, Southern blotting, and gene sequencing. Exotoxin preparation derived from ApxIA336 was analyzed for its bioactivity towards porcine erythrocytes and alveolar macrophages. Analysis results indicated that ApxIA336 contained a kanamycin-resistant cassette inserted immediately after 1005 bp of the apxIA gene. Phenotype analysis of ApxIA336 revealed no difference in the growth rate as compared to the parental strain. Meanwhile, ApxI production was abolished in the bacterial culture supernatant, i.e. exotoxin preparation. The inability of ApxIA336 to produce ApxI corresponded to the loss of hemolytic and cytotoxic bioactivity in exotoxin preparation, as demonstrated by hemolysis, lactate dehydrogenase release, mitochondrial activity, and apoptosis assays. Additionally, the virulence of ApxIA336 appeared to be attenuated by 15-fold in BALB/c mice. Collectively, ApxI, but not other components in the exotoxin preparation of A. pleuropneumoniae serotype 10, was responsible for the hemolytic and cytotoxic effects on porcine erythrocytes and alveolar macrophages.
Actinobacillus pleuropneumoniae/genetics/*pathogenicity/*physiology ; Animals ; *Apoptosis ; Bacterial Proteins/genetics/metabolism ; Blotting, Southern ; Exotoxins/*genetics ; Hemolysin Proteins/genetics/metabolism ; *Hemolysis ; Macrophages, Alveolar/metabolism/*microbiology ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Swine ; Virulence

cry1Ah1, one of holo-type cry genes, cloned in this laboratory from Bacillus thuringiensis strain has been patented in China, and it encoded a protein with strong insecticidal activity against certain lepidopteran insect pests, such as Chilo suppressalis. cry1Ah1 gene is exhibiting good application prospects. In order to improve the expression level of cry1Ah1 gene in rice, and investigate the effect of codon usage preference of gene expression, we designed five different optimized schemes for cry1Ah1 insecticidal critical fragment in accordance with bias of rice codon, to improve G+C content, removed the shear signal and unstable factors. Optimized cry1Ah1 genes were transformed into Escherichia coli Rosetta (DE3) respectively, and 65 kDa polypeptides was expressed normally in inclusion body separately. All of these expressed polypeptides showed insecticidal activity against 2nd-instar larvae of Plutella xylostella and neonate of Chilo suppressalis. After transformation with modified cry1Ah1 genes into Var nippobare, the transgenic rice seedlings were detected by PCR, the positive rate containing target gene was more than 87%. Afterwards, the results of real-time RT-PCR and ELISA assay indicated that the highest expression level of five modified cry1Ah1 genes was that using the highest frequent codons. Average expression amount of Cry1Ah1 polypeptides was 0.104% of total soluble proteins from the positive transgenic rice.
Animals ; Bacillus thuringiensis ; genetics ; metabolism ; Bacterial Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Codon ; genetics ; Endotoxins ; biosynthesis ; genetics ; Hemolysin Proteins ; biosynthesis ; genetics ; Insecticides ; Lepidoptera ; Oryza ; genetics ; Pest Control, Biological ; methods ; Plants, Genetically Modified ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells (BMDCs) and T lymphocytes in vitro.

METHODSAfter BMDCs stimulated by E.coli LLO/OVA, their Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) receptor signalling pathway were examined by superarray hybridization; and the priming effect of the vaccine activated BMDCs on CD4(+)T and CD8(+)T was determined by [3H]thymidine uptake and ELISA, the tumor cytotoxic effect of activated CD8(+)T cells was determined by cytotoxic assay.

RESULTSAfter BMDCs were activated by E. coli LLO/OVA via TLR4, NOD1 receptor and NF-κB signalling pathway, the expression of their surface molecules including MHC class I, MHC class II, CD40, CD80 and CD86 significantly up-regulated; the secretion of IL-12 and IFN-γ increased also. The mature BMDCs stimulated the allergic CD4(+)T and CD8(+)T cells proliferation and their IL-2 and IFN-γ secretion, and the activated CD8(+)T cells effectively killed B16-OVA melanoma cells and RMA-S/OVA lymphoma cells in vitro.

CONCLUSIONE.coli LLO/OVA is effective in inducing BMDCs maturation via activating TLR4 and NOD1 receptor signalling pathway and promoting specific anti-tumor T cell immunity in vitro.

Animals ; Antigens, Neoplasm ; genetics ; pharmacology ; Bacterial Toxins ; genetics ; pharmacology ; Cancer Vaccines ; genetics ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; immunology ; Coculture Techniques ; Cytokines ; immunology ; secretion ; Dendritic Cells ; cytology ; drug effects ; immunology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Female ; Flow Cytometry ; Heat-Shock Proteins ; genetics ; pharmacology ; Hemolysin Proteins ; genetics ; pharmacology ; Immunity, Innate ; drug effects ; Mice ; Mice, Inbred C57BL ; Nod1 Signaling Adaptor Protein ; genetics ; physiology ; Ovalbumin ; genetics ; pharmacology ; Recombinant Fusion Proteins ; genetics ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology ; Toll-Like Receptor 4 ; genetics ; physiology

Cholesterol-dependent cytolysins (CDC) are pore forming toxins. A prototype of the CDC family members is perfringolysin O (PFO), which directly binds to the cell membrane enriched in cholesterol, causing cell lysis. However, an exception of this general observation is intermedilysin (ILY) of Streptococcus intermedius, which requires human CD59 as a receptor in addition to cholesterol for its hemolytic activity. A possible explanation of this functional difference is the conformational variation between the C-terminal domains of the two toxins, particularly in the highly conserved undecapeptide termed tryptophan rich motif. Here, we present the crystal structure of suilysin, a CDC toxin from the infectious swine pathogen Streptococcus suis. Like PFO, suilysin does not require a host receptor for hemolytic activity; yet the crystal structure of suilysin exhibits a similar conformation in the tryptophan rich motif to ILY. This observation suggests that the current view of the structure-function relationship between CDC proteins and membrane association is far from complete.
Amino Acid Sequence ; Animals ; Bacterial Toxins ; chemistry ; Bacteriocins ; chemistry ; Cholesterol ; chemistry ; Crystallography, X-Ray ; Cytotoxins ; chemistry ; Hemolysin Proteins ; chemistry ; genetics ; Molecular Sequence Data ; Point Mutation ; Protein Structure, Tertiary ; Sequence Alignment ; Streptococcus suis ; metabolism ; Swine

Sorghum (Sorghum bicolor L.) was one of the most important crops in the world next to wheat, rice, maize, soybean and barley. Using the callus derived from immature inflorescence as the recipients, we efficiently transformed sorghum varieties 115, ICS21B and 5-27 with the insecticidal Bacillus thuringiensis (Bt) cry1Ab gene carried in the T-DNA of binary vectors which contained hygromycin resistance gene and gus gene via Agrobacterium tumefaciens. After gradient selection with hygromycin, a total of 21 independent transgenic plant lines, 52 transgenic plants were regenerated, and the average stably transformation efficiency was 1.9%. The integration and transcription of cry1Ab gene in transgenic sorghum was confirmed by PCR analysis, Southern blotting and RT-PCR analysis. The Bt proteins were expressed in most transgenic plants with different level from plant to plant by Western blotting and ELISA assay. According to insect bioassay in laboratory, the transgenic plants with a relatively high level of Bt gene expression displayed insect-resistance to pink rice borer (Sesamina inferens).
Agrobacterium tumefaciens ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Endotoxins ; genetics ; metabolism ; Hemolysin Proteins ; genetics ; metabolism ; Pest Control, Biological ; Plants, Genetically Modified ; genetics ; Sorghum ; genetics ; Transformation, Genetic

According to the GenBank sequences (GenBank Accession No. AF539467), one pair of primers was designed to amplify hly gene of Aeromonas hydrophila by PCR. After sequencing, homology analysis indicated that a DNA fragment of 1485 bp was amplified from isolated DNA from Aeromonas hydrophila, and it shared more than 99% homology in nucleotide sequence compared with other reference strains in GenBank. The gene was cloned in pET-28a vector to construct a recombinant plasmid pET-28a-hly, which was transformed into Escherichia coli BL21 (DE3), and the recombinant strain BL21(DE3)(pET-28a-hly) was obtained. The hemolysin was highly expressed when the recombinant strain BL21 (DE3) (pET-28a-hly) was induced by IPTG. The expressed protein was 56 kD as estimated by 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The immunogenicity of the expressed Hly protein was confirmed by Western blotting. Mice were immunized with inactivated whole bacteria vaccine and the genetic engineering vaccines showing promise that all these vaccines have a high protective ability. The results showed that the recombinant strain BL21 (DE3)(pET-28a-hly) could be candidate of hemolysin toxoid vaccine to provide protective immunity against diseases caused by Aeromonas hydrophila.
Aeromonas hydrophila ; genetics ; Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Female ; Hemolysin Proteins ; genetics ; metabolism ; Immunization ; Mice ; Molecular Sequence Data ; Random Allocation ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Sequence Homology ; Toxoids ; immunology ; Vaccines, Synthetic ; immunology