Dengue Envelope-2, Insect
Description
Production and Purification
Expression System:
Purification:
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Proprietary chromatographic techniques achieve >95% purity .
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Sucrose gradient centrifugation (15–60%) isolates mature viral particles .
Formulation:
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Stabilized in 1× PBS, pH 7.4, with 2.5% D-trehalose, protease inhibitors (leupeptin, aprotinin), and 0.099% thimerosal .
Functional Role in Viral Pathogenesis
The E protein mediates host cell entry via receptor binding and membrane fusion:
Table 2: Key Functional Interactions
EPrRec knockdown in Aedes aegypti reduces midgut infection prevalence by 60–80%, confirming its role as a critical receptor . Compound 6 inhibits DENV-2 by trapping virions in endosomal vesicles .
Research Applications
Table 3: Key Studies and Findings
Antigenic Properties
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Domain III (ED3): Contains type-specific (K305, P384) and cross-reactive (K310) epitopes critical for antibody neutralization .
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Mutations (e.g., K310A) disrupt antibody binding but may compromise viral viability .
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Insect-derived E proteins exhibit conformational flexibility in the N-terminal region, affecting antibody recognition .
Challenges and Limitations
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Stability Issues: Low pH induces conformational changes in the fusion loop (FL), complicating structural studies .
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Glycosylation Variability: Insect cell glycosylation differs from human systems, potentially altering immunogenicity .
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Essential Gene Constraint: EPrRec is essential in Aedes aegypti, limiting homozygous knockouts .
Future Directions
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Thermostable Variants: Engineered dimers (e.g., SC.10) could improve vaccine stability .
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Dual-Target Inhibitors: Compounds blocking EPrRec-E protein and Hsc70-3 interactions may prevent mosquito transmission .
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Glycoengineering: Modifying N-glycosylation sites to mimic human patterns for vaccine optimization .
Product Specs
Q&A
FAQs for Researchers on Dengue Virus Envelope Protein 2 (DENV2 E) in Insect Systems
How is the DENV2 E protein receptor identified in Aedes aegypti midgut epithelial cells?
Methodological Approach:
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Receptor Screening: DENV2 virions pretreated with trypsin were incubated with midgut protein extracts. A single 31 kDa protein (AAEL011180, termed EPrRec) was isolated using protein mass spectrometry .
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Validation:
Key Data:
What methods are used to analyze DENV2 E protein interactions with insect-derived receptors?
Experimental Design:
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Virus Overlay Protein-Binding Assay (VOPBA): Used to confirm interactions between DENV2 virions and mucin proteins in Ae. aegypti midguts .
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In Vitro Pull-Down Assays: Recombinant EPrRec immobilized on beads bound DENV2 particles, validated via immunoblotting .
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Surface Plasmon Resonance (SPR): Quantified kinetic binding parameters between EPrRec and DENV2 E glycoprotein .
Critical Consideration:
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False positives in computational interaction predictions (e.g., Drosophila ortholog-based models) require orthogonal validation .
How is recombinant DENV2 E protein expressed and purified in insect systems?
Protocol:
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Baculovirus Expression: Full-length DENV2 E protein forms multimeric aggregates in insect cells, enabling ultracentrifugation-based purification .
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Thermostability Optimization: Mutations (e.g., T76R, K204E) enhance yield (>50-fold increase) and dimerization stability at low concentrations .
Quality Control:
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Immunoblotting with conformation-specific monoclonal antibodies ensures native epitope preservation .
How do conflicting data on DENV2 midgut receptor candidates (e.g., EPrRec vs. mucin) impact experimental design?
Contradiction Analysis:
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EPrRec: Silencing reduces midgut infection prevalence by >50% .
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Mucin: Antibody blocking reduces viral titers but does not fully inhibit infection .
Resolution Strategy:
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Use combinatorial knockdowns (EPrRec + mucin) to assess additive/synergistic effects.
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Compare binding kinetics (e.g., SPR for EPrRec vs. VOPBA for mucin) .
Data Comparison:
| Receptor | Binding Assay | Functional Impact |
|---|---|---|
| EPrRec | SPR, RNAi | 50% infection reduction |
| Mucin | VOPBA, antibody blocking | Partial titer reduction |
What challenges arise in characterizing DENV2 E protein glycosylation in insect cells?
Methodological Limitations:
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Heterogeneity: Insect cell-derived DENV2 E exhibits diverse N-glycans (high-mannose, galactosylated) .
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Analytical Tools: Integrated lectin microarrays and MALDI-TOF-MS resolve glycan structures but require high-purity virus isolates .
Key Findings:
| Glycan Type | Abundance | Functional Role |
|---|---|---|
| High-mannose | 60% | DC-SIGN receptor binding |
| Sialylated | 15% | Immune evasion |
How do partial EPrRec knockdowns (ΔEPrRec +/−) affect DENV2 transmission studies?
Experimental Constraints:
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EPrRec is essential in Ae. aegypti, limiting studies to heterozygotes .
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Workaround: Systemic dsRNA injections further reduce mRNA levels in ΔEPrRec +/− midguts, achieving ~70% infection suppression .
Implications:
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Gene-editing tools (e.g., CRISPR/Cas9) require conditional knockout strategies to bypass lethality.
Table 1: Comparison of DENV2 Interaction Validation Techniques
Product Science Overview
Dengue virus (DENV) is a significant global health concern, causing millions of infections annually. Among the four serotypes of DENV, Dengue Virus Subtype 2 (DENV-2) is particularly notable for its widespread transmission and severe disease manifestations. The development of recombinant DENV-2 using insect cells has emerged as a promising approach for studying the virus and developing vaccines.
DENV is a single-stranded, positive-sense RNA virus belonging to the genus Flavivirus within the family Flaviviridae . The virus is primarily transmitted to humans through the bite of infected Aedes mosquitoes, particularly Aedes aegypti . DENV infections can range from mild dengue fever to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), which can be fatal .
Recombinant DENV-2 refers to genetically engineered versions of the virus, created to study its properties or develop vaccines. These recombinant viruses are often produced using insect cell systems, such as the Aedes aegypti Aag2 cell line . The use of insect cells offers several advantages, including high yield production and the ability to perform post-translational modifications similar to those in the natural mosquito vector .
Insect cell systems, particularly those derived from Aedes mosquitoes, are widely used for the production of recombinant DENV-2. These systems provide a suitable environment for the virus to replicate and express its proteins. The Aedes aegypti Aag2 cell line is commonly used due to its susceptibility to DENV infection and its ability to produce high titers of the virus . Additionally, the C6/36 Aedes albopictus cell line is also utilized for similar purposes .
The development of recombinant DENV-2 using insect cells has several important applications:
- Vaccine Development: Recombinant DENV-2 can be used to create vaccine candidates that elicit strong immune responses. These vaccines can be tested for safety and efficacy in preclinical and clinical trials.
- Virus-Host Interaction Studies: By using recombinant DENV-2, researchers can study the interactions between the virus and its host cells, gaining insights into viral replication, immune evasion, and pathogenesis.
- Drug Screening: Recombinant DENV-2 can be employed in high-throughput screening assays to identify potential antiviral compounds that inhibit viral replication.
