Insecticidal protein genes from Bacillus thuringiensis are currently the most widely used insect-resistant genes. They have been transferred to many crops for breeding and production. Among them, cotton, maize, potato and other insect-resistant crops are commercialized, creating considerable economic benefit. In this review, we summarized advances in identifying functional genes and transgenic crops for insect resistance, compared different strategies for enhancing vigor of insecticidal protein and utilizing gene stacking as well as listing valuable groups of stacked genes. In addition, the methods for multiple gene transformation was discussed.
Animals ; Bacterial Proteins ; genetics ; Crops, Agricultural ; genetics ; Endotoxins ; genetics ; Hemolysin Proteins ; genetics ; Insecta ; Plants, Genetically Modified

To investigate the occurrence and distribution of serotype, specific virulence genes, and pulse field gel electrophoresis (PFGE) patterns in Vibrio parahaemolyticus isolates from Jeonnam, Korea, we tested 87 strains which were identified with V. parahaemolyticus from diarrheal episode patients in 2005. In this study, 16 different O:K serotype combinations of V. parahaemolyticus were determined. The distributions of O and K serotypes were O4:K68 (51.72%), O1:K70 (18.39%), O3:K6 (5.74%), O1:K68 (4.60%) and O3:K57 (4.60%) respectively. Serotype O4:K68 was the regional dominant specific serotype of V. parahaemolyticus in Sinan of Jeonnam, Korea. For the detection of thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh) gene of V. parahaemolyticus, PCR was performed. The tdh gene was detected in all of the V. parahaemolyticus isolates from diarrheal patients, but trh gene was not detected. Analysis of PFGE patterns of 30 V. parahaemolyticus isolates showed 3 groups and 20 types. Among 14 O4:K68 serotypes which were isolated in Sinan, PFGE patterns of 12 strains were closely related (100%), but 2 strains were related by 58.3% and 45.4%, respectively. Also two strains of O1:K4 serotype in Gurye and two strains of O3:K6 serotype in Yeosu were closely related (100%), respectively. Although serotypes (O1:K4, O1:K70, O3:K6 and O4:K68) were different, PFGE patterns were related for more than 80.9%. Therefore, the epidemiological surveillance of V. parahaemolyticus is required by PFGE typing scheme as a further diagnostic tool.
Bacterial Toxins ; Electrophoresis ; Hemolysin Proteins ; Humans ; Korea ; Polymerase Chain Reaction ; Serotyping ; Vibrio ; Vibrio parahaemolyticus

With the rapid development of transgenic technology, the safety of genetically modified products has received extensive attention. Certified reference materials for the detection of genetically modified organisms play important roles in ensuring comparability and traceability of the qualitative and quantitative detection of genetically modified products. However, the development of protein reference materials is relatively slow, and one of the difficulties is the preparation of protein candidates with high purity. The cry1Ah1 gene of Bacillus thuringiensis has been used for the development of transgenic insect-resistant crops because of its excellent insecticidal activity against lepidopteran pests such as Asian corn borer, and has obtained transgenic lines with good insect resistance traits. In order to develop Cry1Ah protein certified reference material, it is urgent to establish a preparation and purification system. In this study, a system for preparing Cry1Ah protein by Bt expression system was optimized, and a high-purity Cry1Ah protein (size exclusion chromatography purity: 99.6%) was obtained by ion-exchange chromatography and size exclusion chromatography stepwise purification. The results of biological activity assay showed that there was no significant difference in the insecticidal activity of purified Cry1Ah protein and protoxin against diamondback moths (Plutella xylostella). Finally, the amino acid sequence of the activated Cry1Ah protein was determined using Edman degradation and mass spectrometry. In summary, the obtained Cry1Ah pure protein can be used for the development of protein reference materials.
Animals ; Bacillus thuringiensis ; Bacterial Proteins ; Cryptochromes ; metabolism ; Endotoxins ; Hemolysin Proteins ; Moths ; Pest Control, Biological ; Plants, Genetically Modified

Bacillus thuringiensis (Bt) produces Cry toxins that are widely used as insecticides in agriculture and forestry. Receptors are important to elucidate the mode of interaction with Cry toxins and toxicity in lepidopteran insects. Here, we purified the Cry toxin from Bt and identified this toxin by flight mass spectrometry as Cry1Ac, and then recombinantly expressed aminopeptidase N (BmAPN6) and repeat domains of cadherin-like protein (CaLP) of B. mori. Using co-immunoprecipitation (co-IP), Far-Western blotting, and enzyme-linked immunosorbent assays (ELISAs), we identified the interaction between Cry1Ac and BmAPN6. Furthermore, analysis of the cytotoxic activity of Cry1Ac toxin in Sf9 cells showed that BmAPN6 directly interacted with Cry1Ac toxin to induce morphological aberrations and cell lysis. We also used co-IP, Far-Western blotting and ELISAs to analyze the interactions of Cry1Ac with three binding sites corresponding to cadherin repeat (CR) 7 CR11, and CR12 of CaLP. Notably, the three repeat domains were essential Cry1Ac binding components in CaLP. These results indicated that BmAPN6 and CaLP served as a functional receptor involved in Bt Cry1Ac toxin pathogenicity. These findings represent an important advancement in our understanding of the mechanisms of Cry1Ac toxicity and provide promising candidate targets for gene editing to enhance resistance to pathogens and increase the economic value of B. mori.
Animals ; Bacillus thuringiensis ; Bacterial Proteins ; metabolism ; Bombyx ; enzymology ; CD13 Antigens ; metabolism ; Cadherins ; metabolism ; Endotoxins ; metabolism ; Hemolysin Proteins ; metabolism ; Larva

Bacillus cereus belongs to Gram-positive bacteria, which is widely distributed in nature and shows certain pathogenicity. Different B. cereus strains carry different subsets of virulence factors, which directly determine the difference in their pathogenicity. It is therefore important to study the distribution of virulence factors and the biological activity of specific toxins for precise prevention and control of B. cereus infection. In this study, the hemolysin BL triayl was expressed, purified, and characterized. The results showed that the bovine pathogenic B. cereus hemolysin BL could be expressed and purified in the prokaryotic expression system, and the bovine pathogenic B. cereus hemolysin BL showed hemolysis, cytotoxicity, good immunogenicity and certain immune protection in mice. In this study, the recombinant expression of hemolysin BL triayl was achieved, and the biological activity of hemolysin BL of bovine pathogenic ceroid spore was investigated. This study may facilitate further investigating the pathogenic mechanism of B. cereus hemolysin BL and developing a detection method for bovine pathogenic B. cereus disease.
Cattle ; Animals ; Mice ; Bacterial Proteins/metabolism* ; Bacillus cereus/metabolism* ; Hemolysin Proteins/metabolism* ; Virulence Factors/metabolism* ; Enterotoxins/metabolism*

OBJECTIVETo establish a mouse model of humoral immune response by immunization with rabbit red blood cells (RRBCs).

METHODSThe mice were immunized with RRBCs and the serum hemolysin level was measured by micro-hemolysis spectrophotometry.

RESULTSThe peak time needed for hemolysin production against RRBCs was 6 days after the immunization, and 20% RRBCs in a total volume of 0.2 ml was optimal for intraperitoneal injection. Hydrocortisone (25 mg/kg) and cyclophosphamide (20 mg/kg) inhibited hemolysin production. Mannatide (4 mg/kg) produced no significant effect on serum hemolysin level in normal mice, but significantly potentiated hemolysin production in immunosuppressed mice induced by cyclophosphamide (20 mg/kg).

CONCLUSIONIntraperitoneal RRBC injection is feasible for establishing mouse models of humoral immune response.

Animals ; Erythrocytes ; immunology ; Female ; Guinea Pigs ; Hemolysin Proteins ; blood ; Immunity, Humoral ; Immunization ; Male ; Mice ; immunology ; Models, Animal ; Rabbits

Actinobacillus pleuropneumoniae ; drug effects ; genetics ; Animals ; Bacterial Proteins ; genetics ; Blotting, Western ; Drug Resistance, Bacterial ; genetics ; Green Fluorescent Proteins ; genetics ; Hemolysin Proteins ; genetics ; Hemolysis ; Mice ; Mutation ; Polymerase Chain Reaction

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

The alpha-hemolysin protein of Staphylococcus aureus, which was expressed in Escherichia coli BL21 (DE3) with recombinant pET32a(+)-alpha-HL plasmid, was purified with gel filtration chromatography (GFC) and Ni-NTA spin columns. The quality and biological characteristic were compared. First, the purified products were analyzed with SDS-PAGE, and the expected protein band was with a molecular mass of 53 kD. Second, protein concentration was determined by the method of Bradford, and the median hemolytic dose potency (HD50) was finally analyzed with rabbit erythrocyte. The protein purified with GFC was 0.337 mg/mL, its hemolysis activity was 1519 HU/mg, and hemolysin yield was 14.04%. Meanwhile, the protein purified with the Ni-NTA Spin Columns was 0.35 mg/mL, its hemolysis activity was 1463 HU/mg, and hemolysin yield was 17.5%, respectively. The results showed that there is no significant difference in the quality, hemolysis activity and yield of the recombinant proteins purified with Ni-NTA spin columns and GFC.
Chromatography, Gel ; methods ; Escherichia coli ; genetics ; metabolism ; Hemolysin Proteins ; genetics ; isolation & purification ; Nitrilotriacetic Acid ; analogs & derivatives ; Organometallic Compounds ; Recombinant Proteins ; genetics ; isolation & purification ; Staphylococcus aureus ; genetics ; metabolism

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