Transmembrane emp24 domain (TMED) gene is closely related to immune response, signal transduction, growth and disease development in mammals. However, only the Drosophila TMED gene has been reported on insects. We identified the TMED family genes of silkworm, Tribolium castaneum, tobacco moth and Italian bee from their genomes, and found that the TMED family gene composition patterns of one α-class, one β-class, one δ-class and several γ-classes arose in the common ancestor of pre-divergent Hymenoptera insects, while the composition of Drosophila TMED family members has evolved in a unique pattern. Insect TMED family γ-class genes have evolved rapidly, diverging into three separate subclasses, TMED6-like, TMED5-like and TMED3-like. The TMED5-like gene was lost in Hymenoptera, duplicated in the ancestors of Lepidoptera and duplicated in Drosophila. Insect TMED protein not only has typical structural characteristics of TMED, but also has obvious signal peptide. There are seven TMED genes in silkworm, distributed in six chromosomes. One of seven is single exon and others are multi-exons. The complete open reading frame (ORF) sequences of seven TMED genes of silkworm were cloned from larval tissues and registered in GenBank database. BmTMED1, BmTMED2 and BmTMED6 were expressed in all stages and tissues of the silkworm, and all genes were expressed in the 4th and 5th instar and silk gland of the silkworm. The present study revealed the composition pattern of TMED family members, their γ class differentiation and their evolutionary history, providing a basis for further studies on TMED genes in silkworm and other insects.
Animals ; Bombyx/metabolism* ; Genes, Insect/genetics* ; Moths/metabolism* ; Insecta/metabolism* ; Drosophila ; Insect Proteins/metabolism* ; Phylogeny ; Mammals/genetics*

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

Rice stripe virus (RSV) transmitted by the small brown planthopper causes severe rice yield losses in Asian countries. Although viral nuclear entry promotes viral replication in host cells, whether this phenomenon occurs in vector cells remains unknown. Therefore, in this study, we systematically evaluated the presence and roles of RSV in the nuclei of vector insect cells. We observed that the nucleocapsid protein (NP) and viral genomic RNAs were partially transported into vector cell nuclei by utilizing the importin α nuclear transport system. When blocking NP nuclear localization, cytoplasmic RSV accumulation significantly increased. In the vector cell nuclei, NP bound the transcription factor YY1 and affected its positive regulation to FAIM. Subsequently, decreased FAIM expression triggered an antiviral caspase-dependent apoptotic reaction. Our results reveal that viral nuclear entry induces completely different immune effects in vector and host cells, providing new insights into the balance between viral load and the immunity pressure in vector insects.
Animals ; Cell Nucleus ; Hemiptera/metabolism* ; Insect Vectors/genetics* ; Insecta ; Nucleocapsid Proteins/metabolism* ; Oryza ; Plant Diseases ; Tenuivirus/metabolism* ; Virus Replication

Three new phenazine-type compounds, named phenazines SA-SC (1-3), together with four new natural products (4-7), were isolated from the fermentation broth of an earwig-associated Streptomyces sp. NA04227. The structures of these compounds were determined by extensive analyses of NMR, high resolution mass spectroscopic data, as well as single-crystal X-ray diffraction measurement. Sequencing and analysis of the genome data allowed us to identify the gene cluster (spz) and propose a biosynthetic pathway for these phenazine-type compounds. Additionally, compounds 1-5 exhibited moderate inhibitory activity against acetylcholinesterase (AChE), and compound 3 showed antimicrobial activities against Micrococcus luteus.
Animals ; Anti-Bacterial Agents ; chemistry ; metabolism ; pharmacology ; Bacterial Proteins ; genetics ; metabolism ; Crystallography, X-Ray ; Insecta ; microbiology ; Magnetic Resonance Spectroscopy ; Microbial Sensitivity Tests ; Micrococcus luteus ; drug effects ; Molecular Structure ; Multigene Family ; Phenazines ; chemistry ; metabolism ; pharmacology ; Streptomyces ; chemistry ; genetics ; metabolism

As one of the most important aquatic fish, Micropterus salmoides suffers lethal and epidemic disease caused by rhabdovirus at the juvenile stage. In this study, a new strain of M. salmoides rhabdovirus (MSRV) was isolated from Yuhang, Zhejiang Province, China, and named MSRV-YH01. The virus infected the grass carp ovary (GCO) cell line and displayed virion particles with atypical bullet shape, 300-500 nm in length and 100-200 nm in diameter under transmission electron microscopy. The complete genome sequence of this isolate was determined to include 11 526 nucleotides and to encode five classical structural proteins. The construction of the phylogenetic tree indicated that this new isolate is clustered into the Vesiculovirus genus and most closely related to the Siniperca chuatsi rhabdovirus. To explore the potential for a vaccine against MSRV, a glycoprotein (1-458 amino acid residues) of MSRV-YH01 was successfully amplified and cloned into the plasmid pFastBac1. The high-purity recombinant bacmid-glycoprotein was obtained from DH10Bac through screening and identification. Based on polymerase chain reaction (PCR), western blot, and immunofluorescence assay, recombinant virus, including the MSRV-YH01 glycoprotein gene, was produced by transfection of SF9 cells using the pFastBac1-gE2, and then repeatedly amplified to express the glycoprotein protein. We anticipate that this recombinant bacmid system could be used to challenge the silkworm and develop a corresponding oral vaccine for fish.
Animals ; Baculoviridae/metabolism* ; Bass/metabolism* ; Carps/virology* ; Cell Line ; Female ; Genetic Techniques ; Genome, Viral ; Glycoproteins/biosynthesis* ; Insecta ; Ovary/virology* ; Phylogeny ; Plasmids/metabolism* ; Recombinant Proteins/biosynthesis* ; Rhabdoviridae/metabolism*

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

Baculoviruses are a family of arthropod-specific viruses that produce two morphologically distinct types of virions (budded and occlusion-derived) in their lifecycle. Baculoviruses establish infection in the midgut of their host via the oral route: occlusion-derived virions have pivotal roles in these processes. This review summarizes the basic characteristics of baculoviruses, and discusses the composition and classification of baculovirus occlusion-derived virions. The latter focuses mainly on the evolution and role of multiple occlusion-derived virions in the lifecycle of baculoviruses. These achievements should aid understanding the evolution and infection mechanisms of baculoviruses.
Animals ; Baculoviridae ; genetics ; growth & development ; physiology ; Insecta ; virology ; Viral Proteins ; genetics ; metabolism ; Virion ; genetics ; growth & development ; physiology

Eukaryotic membrane proteins, many of which are key players in various biological processes, constitute more than half of the drug targets and represent important candidates for structural studies. In contrast to their physiological significance, only very limited number of eukaryotic membrane protein structures have been obtained due to the technical challenges in the generation of recombinant proteins. In this review, we examine the major recombinant expression systems for eukaryotic membrane proteins and compare their relative advantages and disadvantages. We also attempted to summarize the recent technical strategies in the advancement of eukaryotic membrane protein purification and crystallization.
Animals ; Escherichia coli ; genetics ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; HEK293 Cells ; Humans ; Insecta ; cytology ; genetics ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; metabolism ; Yeasts ; genetics

To prepare recombinant human retinol binding protein 4 (RBP4) by using the baculovirus expression system and to detect its immunogenicity, the fusion DNA fragment of secretory signal peptide SS64 and human RBP4 gene was subcloned into a baculovirus transfer vector pFastBac-dual(pFBd), and the corresponding recombinant transfer plasmid was transformed into E. coli strain DH10bac, after transposition recombinant shuttle bacmid was screened out. The logarithmic phase Sf9 cells were transfected with the recombinant bacmid and then the recombinant baculovirus containing hRBP4 expression box were generated. After amplification of recombinant baculovirus, the recombinant baculovirus seeds were obtained. To express human RBP4, logarithmic phase Sf9 cells were infected with the virus seeds and SDS-PAGE and Western blotting were used to detect and identify the expression. Finally, to prepare a batch of RBP4 protein, logarithmic phase Sf9 cells in suspension culture were infected with recombinant baculovirus seeds and the supernatant was harvested after 120 hours post-infection for purification. Finally for preparation of polyclonal antibody and evaluation of immunogenicity, the recombinant hRBP4 from insect cells and from E. coli were immunized rabbits. Restriction enzyme digestion and sequencing confirmed that the recombinant baculovirus transfer plasmid was constructed correctly, and subsequently recombinant RBP4-bacmid was generated successfully. SDS-PAGE and Western blotting analysis suggested that human RBP4 protein was highly expressed in Sf9 cells with the molecular weight of approximately 23 kDa. The recombinant RBP4 protein could be secreted into the medium efficiently, and the expression level was calculated amount of 100 mg/L. Finally the rabbit antiserum was harvested after recombinant RBP4 immunization, therein the titer of antiserum against baculovirus recombinant RBP4 is 1:100 000 whereas the titer of antiserum against E. coli recombinant RBP4 is only 1:10 000. Overall, human RBP4 was high efficiently expressed successfully with good antigenicity in baculovirus system, and high affinity antiserum was obtained. A solid foundation was laid for the next step of the preparation of human serum RBP4 detection kit.
Animals ; Baculoviridae ; genetics ; Blotting, Western ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Genetic Vectors ; Humans ; Immune Sera ; Insecta ; Rabbits ; Recombinant Proteins ; biosynthesis ; immunology ; Retinol-Binding Proteins, Plasma ; biosynthesis ; immunology ; Sf9 Cells ; metabolism ; Transfection

To investigate a baculovirus insect cell system for expressing an interferon alpha 2b (IFNa2b)/immunoglobulin G-4 (IgG4) Fc fusion protein, which has long-acting antiviral effects. Human IFNa2b and IgG4 Fc cDNAs were generated by molecular cloning and inserted into a baculovirus shuttle vector, which was then transposed into the DH10 Bac strain to form recombinant Bacmid-IFN/Fc. The Bacmid-IFN/Fc was transfected into High five insect cells, and expression of the IFN/Fc fusion protein was detected by Western blotting and its biological activity was assessed by the cytopathic effect inhibition method. The IFNa2b and IgG4 Fc cDNA fragments were successfully amplified by RT-PCR using human peripheral lymphocytes. After cloning into the baculovirus shuttle vector, pFastBac1, and transforming into DH10 Bac competent cells, screening identified positive clones carrying the recombinant Bacmid-IFN/Fc. A Bacmid-IFN/Fc clone was successfully transfected into the High five insect cells and packaged into the baculovirus for expression of the IFN/Fc fusion protein. Western blotting revealed that the fusion protein expression was specific, and yielded a protein of 45 kD in size. The in vitro antiviral activity of the IFN/Fc fusion protein was 580 IU/mL. A novel IFN/Fc fusion protein was successfully generated using a baculovirus insect cell system, which may prove useful for providing future experimental data for development of a new long-acting interferon to treat chronic viral hepatitis.
Animals ; Antiviral Agents ; metabolism ; Baculoviridae ; genetics ; Cell Line ; Cloning, Molecular ; Gene Expression ; Gene Fusion ; Genetic Vectors ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Insecta ; Interferon-alpha ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection