Recombinant Renibacterium salmoninarum Elongation factor Tu (tuf)
Description
Elongation Factor Tu (EF-Tu): Canonical and Moonlighting Functions
EF-Tu is a highly conserved GTP-binding protein critical for bacterial translation, facilitating aminoacyl-tRNA delivery to ribosomes . Beyond its core role, EF-Tu exhibits moonlighting functions in pathogens, including:
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Adhesion: Binding to extracellular matrix components (e.g., collagen, fibronectin) for host cell attachment .
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Immune modulation: Interacting with host receptors to evade recognition or suppress immune responses .
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Biofilm formation: Contributing to structural integrity in biofilms (e.g., Gallibacterium anatis) .
EF-Tu in Renibacterium salmoninarum: Current Knowledge Gaps
While R. salmoninarum is a major salmonid pathogen, its EF-Tu has not been extensively characterized. Key observations include:
Molecular Insights into R. salmoninarum
Although EF-Tu research is limited, studies on R. salmoninarum highlight other virulence factors:
Research Opportunities for Recombinant R. salmoninarum EF-Tu
Future studies could explore:
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Structural analysis: Identify surface-exposed motifs (e.g., SLiMs) enabling moonlighting functions .
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Vaccine development: Test rEF-Tu as a subunit vaccine, leveraging its immunogenic potential in other pathogens .
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Gene knockout models: Assess EF-Tu’s role in translation and pathogenesis via tuf deletion mutants.
Product Specs
Target Background
KEGG: rsa:RSal33209_2171
STRING: 288705.RSal33209_2171
Q&A
Experimental Design for Studying Recombinant Renibacterium salmoninarum Elongation Factor Tu (tuf)
Q: What experimental design strategies can be employed to study the recombinant elongation factor Tu (tuf) in Renibacterium salmoninarum?
A: To study recombinant Renibacterium salmoninarum elongation factor Tu (tuf), researchers can employ several experimental design strategies:
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Cloning and Expression: Clone the tuf gene into an appropriate expression vector and transform it into a suitable host organism, such as Escherichia coli, to produce recombinant protein.
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Protein Purification: Use techniques like affinity chromatography to purify the recombinant protein for further analysis.
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Functional Assays: Conduct assays to assess the protein's role in protein synthesis and its potential as a vaccine candidate or diagnostic marker.
Data Analysis and Contradiction Resolution
Q: How can researchers resolve data contradictions when analyzing the expression levels of recombinant Renibacterium salmoninarum elongation factor Tu (tuf) in different hosts?
A: Resolving data contradictions involves several steps:
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Replication: Repeat experiments to ensure consistency.
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Normalization: Normalize data using appropriate controls to account for variations.
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Statistical Analysis: Use statistical methods to identify significant differences and trends.
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Literature Review: Compare findings with existing literature to contextualize results.
Advanced Research Questions: Immunogenicity and Vaccine Development
Q: What are the key considerations for investigating the immunogenicity of recombinant Renibacterium salmoninarum elongation factor Tu (tuf) as a potential vaccine candidate?
A: When investigating the immunogenicity of recombinant Renibacterium salmoninarum elongation factor Tu (tuf) for vaccine development:
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Immunization Protocols: Design immunization protocols to assess immune responses in model organisms.
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Antibody Titers: Measure antibody titers to evaluate humoral immunity.
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Cellular Immunity: Assess cellular immune responses using techniques like ELISPOT assays.
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Challenge Studies: Conduct challenge studies to evaluate vaccine efficacy against Renibacterium salmoninarum infection.
Methodological Considerations for Real-Time PCR
Q: How can researchers optimize real-time PCR conditions for detecting and quantifying Renibacterium salmoninarum DNA, focusing on the tuf gene?
A: Optimizing real-time PCR for detecting Renibacterium salmoninarum DNA involves:
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Primer Design: Design specific primers targeting the tuf gene.
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Probe Selection: Use TaqMan probes for specificity and sensitivity.
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Optimization of Cycling Conditions: Adjust annealing temperatures and cycle numbers to improve specificity and efficiency.
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Internal Controls: Include internal controls to monitor PCR efficiency and detect potential inhibitors.
Comparative Analysis of Different Detection Methods
Q: What are the advantages and limitations of using real-time PCR versus nested PCR for detecting Renibacterium salmoninarum?
A:
| Method | Advantages | Limitations |
|---|---|---|
| Real-time PCR | High sensitivity, specificity, and speed; quantitative capabilities | Requires specific equipment and expertise; potential for inhibition |
| Nested PCR | High sensitivity; can be more specific with nested primers | Time-consuming; risk of contamination; not quantitative |
Real-time PCR offers rapid quantification, while nested PCR provides high sensitivity but is more labor-intensive.
Advanced Techniques for Protein Expression and Purification
Q: What advanced techniques can be used to enhance the expression and purification of recombinant Renibacterium salmoninarum elongation factor Tu (tuf)?
A: Advanced techniques for enhancing expression and purification include:
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Optimization of Expression Conditions: Adjust temperature, pH, and inducer concentration to maximize protein yield.
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Use of Fusion Tags: Employ fusion tags like His-tag or GST for efficient purification via affinity chromatography.
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Protein Refolding: Implement refolding protocols if the protein is expressed in inclusion bodies.
Bioinformatics Tools for Sequence Analysis
Q: What bioinformatics tools can be used to analyze the sequence and structure of Renibacterium salmoninarum elongation factor Tu (tuf)?
A: Bioinformatics tools for sequence and structure analysis include:
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BLAST: For sequence similarity searches.
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ClustalW: For multiple sequence alignment.
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SWISS-MODEL: For protein structure prediction.
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Phyre2: For protein structure prediction and functional site identification.
These tools help in understanding the evolutionary conservation and functional sites of the protein.
Challenges in Studying Recombinant Proteins in Aquatic Pathogens
Q: What are the challenges faced when studying recombinant proteins like elongation factor Tu (tuf) in aquatic pathogens like Renibacterium salmoninarum?
A: Challenges include:
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Cultivation Difficulty: Renibacterium salmoninarum is fastidious and difficult to culture.
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Limited Genetic Tools: Few genetic manipulation tools are available for this bacterium.
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Host-Pathogen Interaction Complexity: Studying host-pathogen interactions requires complex experimental setups.
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Environmental Factors: Aquatic environments can introduce variability in experimental conditions.
Future Directions in Vaccine Development
Q: What future directions should researchers consider when developing vaccines based on recombinant Renibacterium salmoninarum elongation factor Tu (tuf)?
A: Future directions include:
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Multivalent Vaccines: Developing vaccines that target multiple antigens.
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Adjuvant Selection: Identifying optimal adjuvants to enhance immune responses.
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Delivery Systems: Exploring novel delivery systems, such as nanoparticles or viral vectors.
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Field Trials: Conducting field trials to assess vaccine efficacy in natural settings.
