RESUMEN
Baculovirus expression vector systems (BEVS) are widely used to produce heterologous proteins for a wide range of applications. Developed more than 30 years ago, BEVS have been constantly modified to improve product quality and ease-of-use. Plasmid reagents were tailored and engineered to facilitate introduction of heterologous genes into baculoviral genomes. At the same time, detrimental modalities such as genes encoding proteases or apoptotic factors were removed to improve protein yield. Advances in DNA synthesis and manipulation now enable the engineering of part or whole synthetic baculovirus genomes, opening up new avenues to redesign and tailor the system to specific applications. Here, we describe a simple protocol for designing and constructing baculovirus genomes comprising segments of synthetic DNA through the use of iterative Red/ET homologous recombination reactions.
Asunto(s)
Baculoviridae/genética , Biotecnología/métodos , Vectores Genéticos , Cromosomas Artificiales Bacterianos/genética , Ingeniería Genética , Genoma Viral , Recombinación Homóloga , Plásmidos , Biología Sintética/métodosRESUMEN
Bombyx mori nucleopolyhedrosis virus (BmNPV) is a highly pathogenic virus to domestic silkworm Bombyx mori, often causing severe economic losses to silk industry. Interestingly, there are no documented reports of NPV infection in the wild silkworm Antheraea mylitta. Analysis of gene expression datasets and comparative genomic analysis of sequence datasets from B. mori and A. mylitta was undertaken to unravel the potential immune-related proteins and immune pathways involved. The B. mori silkworm races Sarupat and CSR-2 which are resistant and susceptible to BmNPV respectively were selected and challenged with virus to study BmNPV resistance related genes and their expression profile. The genes were filtered to isolate membrane proteins, their sequences were retrieved from UniProt and were compared against A. mylitta using BLASTp to search for similarity. Major proteins were putative defence proteins. Further, KEGG database was used to check for the presence of differentially regulated proteins in certain metabolic pathways. The analysis of the pathways was carried out using cytoscape software based on betweeness centrality and stress. The PI3K-Akt pathway was found to be the hub of all signals triggered during the course of NPV infection. We analyzed how the NPV infection modulates PI3K-Akt signaling by gene expression studies of the key regulator of the pathway i.e. Akt using real-time PCR in A. mylitta. A significant upregulation of Akt expression from 72 h post infection reaching its peak with a 2.30 fold change at 120 h pi clearly indicates an enhanced level of immune response in host towards the viral infection.
RESUMEN
RNA interference (RNAi) has become an efficient tool for inducing resistance to viruses in many organisms. In this study, Escherichia coli cells were engineered to produce stable double-stranded RNA (dsRNA) against the nucleopolyhedrosis virus to elicit RNAi in silkworms. The immediate-early-1 (ie-1) and late expression factor-1 (lef-1) genes of the Bombyx mori nucleopolyhedrovirus (BmNPV) involved in viral DNA multiplication were cloned in the plasmid L4440 under the influence of the double T7 promoter and transformed to E. coli HT115 DE3 host cells. On induction with isopropyl ß-D-thiogalactopyranoside, these cells efficiently produced dsRNA of the cloned genes. The B. mori larvae were fed with 50 µL of E. coli cells expressing ie-1 and lef-1 dsRNAs (each approximately 25 µg) to elicit RNAi. The semi-quantitative and quantitative PCR analysis of RNA from the midgut of the dsRNA-fed larvae revealed a significant reduction in the expression of the target genes involved in BmNPV multiplication, which restricted virus copy numbers to 100 compared with 1.9 × 105 in the infected controls. Furthermore, the dsRNA-fed infected larvae showed > 50% increased survivability compared with the infected controls. The study revealed the successful use of bacteria as vectors for efficiently delivering dsRNA to elicit RNAi against BmNPV in silkworms.