RESUMEN
The baculovirus expression vector system is one of the most powerful and versatile eukaryotic expression systems available. However, as the recombinant baculovirus is usually generated by replacing the foreign gene into the polyhedrin locus, the resulting polyhedrin-negative virus is less infectious to the host larvae when administered via oral ingestion. This limits the large-scale production of the recombinant protein, as the host larvae can only be inoculated through dorsal injection, which is a laborious task. In this paper, we describe a new Bombyx mori nucleopolyhedrovirus polyhedrin-plus Bac-to-Bac baculovirus expression system for application in silkworm, B. mori. In this system, the foreign gene and the polyhedrin are co-expressed, and polyhedra are produced as in the wild-type virus, and thus the recombinant baculovirus can be used directly via oral infection. It effectively improves the efficiency of the baculovirus expression system and also widens the application of baculovirus in other fields, such as the development of new biological insecticides.
Asunto(s)
Bombyx/genética , Expresión Génica , Vectores Genéticos/genética , Nucleopoliedrovirus/genética , Transducción Genética/métodos , Proteínas Estructurales Virales/genética , Animales , Bombyx/metabolismo , Bombyx/virología , Vectores Genéticos/metabolismo , Larva/genética , Larva/metabolismo , Larva/virología , Nucleopoliedrovirus/metabolismo , Proteínas de la Matriz de Cuerpos de Oclusión , Proteínas Estructurales Virales/metabolismoRESUMEN
The cDNA encoding an antiviral protein SP-2 against BmNPV was cloned from the midgut of Chinese wild silkworm, Bombyx mandarina Moore (GenBank access AY945210) based on the available information of the domesticated silkworm. Its cDNA was 855 bp encoding 284 amino acids with predicted molecular weight of 29.6 kDa. Its full length in genomics was 1376 bp, including 5 exons and 4 introns. The expression analysis indicated that it was only expressed in midgut, and its expression level was higher during feeding stage of larval instars while very lower during the moltism and mature stages. The deduced amino acid sequence of this protein showed eight-amino-acid variation compared with the counterpart of domesticated silkworm. Its antiviral activity was assayed through in vitro test. The results indicated that it showed strong bioactivity against BmNPV, and its activity was 1.6 fold higher that the counterpart of domesticated silkworm.
Asunto(s)
Antivirales/metabolismo , Bombyx/metabolismo , Proteínas de Insectos/metabolismo , Secuencia de Aminoácidos , Animales , Antivirales/química , Bombyx/química , Bombyx/genética , China , Clonación Molecular , Regulación de la Expresión Génica , Genoma/genética , Proteínas de Insectos/química , Proteínas de Insectos/genética , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
The Bombyx mori nucleopolyhedrovirus (BmNPV) odv-e56 gene is a late gene and encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. To determine its role in the BmNPV life cycle, an odv-e56 null virus, BmE56D, was constructed through homologous recombination. A repaired virus was also constructed, named BmE56DR. The production of budded virion (BV) and polyhedra, the replication of viral DNA, and the morphological of infected BmN cells were analyzed, revealing no significant difference among the BmE56D, the wild-type (WT), and the BmE56DR virus. Larval bioassays demonstrated that injection of BmE56D BV into the hemocoel could kill B. mori larvae as efficiently as repaired and WT viruses, however BmE56D was unable to infect the B. mori larvae when inoculated per os. Thus, these results indicated that ODV-E56 envelope protein of BmNPV is also a per os infectivity factor (PIF), but is not essential for virus replication.
Asunto(s)
Bombyx/virología , Nucleopoliedrovirus/patogenicidad , Proteínas del Envoltorio Viral/metabolismo , Factores de Virulencia/metabolismo , Animales , Prueba de Complementación Genética , Larva/virología , Nucleopoliedrovirus/genética , Recombinación Genética , Análisis de Supervivencia , Proteínas del Envoltorio Viral/deficiencia , Virulencia , Factores de Virulencia/deficiencia , Replicación ViralRESUMEN
A cell line is an important experimental platform for biological sciences as it can basically reflect the biology of its original organism. In this study, we firstly characterized the proteome of cultured BmN cells, derived from Bombyx mori. Total 1478 proteins were identified with two or more peptides by using 1D (one-dimensional) SDS/PAGE and LTQ-Orbitrap. According to the gene ontology annotation, these proteins presented diverse pI values and molecular masses, involved in various molecular functions, including catalytic activity, binding, molecular transducer activity, motor activity, transcription regulator activity, enzyme regulator activity and antioxidant activity. Some proteins related to virus infection were also identified. These results provided us with useful information to understand the molecular mechanism of B. mori as well as antiviral immunity.
Asunto(s)
Bombyx/metabolismo , Proteínas de Insectos/química , Proteoma/química , Proteómica/métodos , Animales , Línea Celular , Proteínas de Insectos/análisis , Proteoma/análisisRESUMEN
The midgut is the digestive apparatus of the silkworm and its proteome was studied by using nano-LC (liquid chromatography) electrospray ionization MS/MS (tandem MS). MS data were analysed by using X!Tandem searching software using different parameters and validated by using the Poisson model. A total of 90 proteins were identified and 79 proteins were described for the first time. Among the new proteins, (i) 22 proteins were closely related to the digestive function of the midgut, including 11 proteins of digestive enzymes secreted by the epithelium, eight proteins of intestine wall muscle and mechanical digestion and three proteins of peritrophic membrane that could prevent the epithelium from being mechanically rubbed; (ii) 44 proteins were involved in metabolism of substance and energy; and (iii) 11 proteins were associated with signal transduction, substance transport and cell skeleton.