Recombinant Rabbit Tumor necrosis factor (TNF), partial (Active)

What is the structure and function of rabbit TNF in comparison to other species?

Rabbit TNF, like mouse TNF-alpha, functions as a pleiotropic molecule central to inflammation and immune responses. While specific rabbit TNF structures may vary slightly, the general structure includes an extracellular domain (ECD), a transmembrane segment, and a cytoplasmic domain. Mouse TNF-alpha, for example, consists of a 35 amino acid cytoplasmic domain, a 21 amino acid transmembrane segment, and a 179 amino acid ECD .

TNF assembles intracellularly to form a noncovalently linked homotrimer expressed on the cell surface. After shedding by TACE/ADAM17, the released bioactive cytokine forms a 55 kDa soluble trimer of the TNF extracellular domain . Cross-species reactivity studies have demonstrated that murine TNF-α maintains biological activity in rabbits, suggesting structural and functional conservation across species .

How should recombinant rabbit TNF be reconstituted and stored?

Based on standard protocols for recombinant TNF proteins, lyophilized recombinant rabbit TNF should be reconstituted under sterile conditions. For carrier-free formulations, reconstitute in sterile PBS at a concentration of approximately 100 μg/mL. For formulations containing bovine serum albumin (BSA) as a carrier protein, reconstitute in sterile PBS containing at least 0.1% human or bovine serum albumin .

For optimal stability:

• Store lyophilized protein at -20°C to -80°C

• After reconstitution, prepare working aliquots and store at -20°C to -80°C

• Avoid repeated freeze-thaw cycles by using a manual defrost freezer

• Use reconstituted protein within 1-3 months when stored properly

What are the primary applications of recombinant rabbit TNF in research?

Recombinant rabbit TNF has several important research applications:

• In vitro bioassays: Investigating cellular responses to inflammatory stimuli

• Ex vivo tissue studies: Examining TNF effects on tissue explants

• Animal models of inflammation: Particularly in neuroinflammation, arthritis, and sepsis models

• Receptor binding studies: Analyzing TNF-receptor interactions

• Antibody production: Generating anti-rabbit TNF antibodies for research

Notably, TNF plays critical roles in central nervous system inflammation. In experimental models of tuberculous meningitis, TNF levels in cerebrospinal fluid correlate with disease severity and pathogenesis .

How does rabbit TNF compare to mouse and human TNF in terms of biological activity and cross-reactivity?

Cross-species reactivity is a critical consideration when selecting TNF for experiments. Evidence shows that murine TNF-α maintains biological activity in rabbits. In intrathecal injection studies, 4 μg of recombinant murine TNF-α induced significant biological responses in rabbits, confirming cross-species activity .

Among different species, TNF maintains significant sequence homology. For example, within the extracellular domain, mouse TNF-alpha shares 94% amino acid sequence identity with rat and 70%-77% with bovine, canine, cotton rat, equine, feline, human, porcine, and rhesus TNF-alpha . This conservation often translates to functional cross-reactivity, making mouse TNF suitable for some rabbit experimental systems.

When designing experiments:

• Test dose-response relationships if using TNF from another species in rabbit systems

• Validate biological activity using species-appropriate positive controls

• Consider species-specific differences when interpreting results

What methodologies can effectively measure recombinant rabbit TNF activity in experimental systems?

Several methodologies are available for measuring TNF activity:

When measuring TNF in cerebrospinal fluid samples, researchers should use validated ELISA kits with appropriate sensitivity ranges, as demonstrated in experimental meningitis studies .

How does partial recombinant rabbit TNF differ from full-length in terms of biological activity?

Partial recombinant proteins contain only specific segments of the full protein. For TNF, the biologically active domain typically resides in the extracellular portion. In mouse TNF-alpha, for example, the amino acid region 80-235 contains the functional domain .

Key differences include:

• Receptor binding: Partial TNF proteins maintain receptor binding capacity if they contain the receptor-binding domain.

• Potency: Partial proteins may exhibit altered potency compared to full-length proteins. For example, mouse TNF-alpha (aa 80-235) retains biological activity with an ED50 of 8-50 pg/mL in appropriate bioassays .

• Stability: Partial proteins often demonstrate increased stability compared to full-length versions.

• Specificity: Some partial proteins show modified receptor specificity or altered downstream signaling.

When using partial recombinant rabbit TNF, researchers should validate its biological activity in their specific experimental system before proceeding with full studies.

What are the optimal experimental conditions for using recombinant rabbit TNF in neuroinflammation studies?

When studying neuroinflammation using recombinant rabbit TNF:

• Dosage determination: Titrate concentrations based on your experimental system. In rabbit models of CNS infection, significant biological effects were observed with doses in the μg range for intrathecal administration .

• Administration routes:

  • Intrathecal: Direct CNS delivery (typically 1-5 μg)

  • Intracerebral: Localized brain delivery

  • Systemic: For peripheral inflammation models

• Timing considerations: Peak TNF responses in cerebrospinal fluid typically occur within 2 hours post-stimulation, with significant levels persisting for up to 8 days in infection models .

• Readout parameters:

  • Leukocyte accumulation in CSF (counts typically range from hundreds to >15,000 cells/mm³)

  • CSF protein levels (indicates blood-brain barrier integrity)

  • Histopathological assessment of meningeal inflammation

  • Behavioral changes

  • Downstream cytokine production

How should controls be designed for experiments using recombinant rabbit TNF?

Proper controls are essential for rigorous TNF research:

• Vehicle controls: Include vehicle-only (PBS or PBS+BSA) treated samples to account for potential effects of the reconstitution buffer.

• Heat-inactivated controls: Heat-denatured TNF (typically 95°C for 10 minutes) to confirm that observed effects are due to the biological activity of the protein rather than contaminants.

• Blocking controls: Co-administration of TNF with neutralizing antibodies or soluble TNF receptors to confirm specificity.

• Dose-response analysis: Include multiple concentrations of TNF to establish dose-dependence of observed effects.

• Time course controls: Sample collection at multiple time points to capture the kinetics of TNF-induced responses, particularly important as TNF effects in CSF show time-dependent patterns .

What techniques can effectively quantify rabbit TNF-induced inflammation in central nervous system models?

Quantification of TNF-induced CNS inflammation utilizes multiple complementary techniques:

• CSF analysis:

  • Leukocyte count and differential (typically showing >90% mononuclear cells in mycobacterial CNF infection models)

  • Protein concentration (indicator of blood-brain barrier integrity)

  • Cytokine/chemokine profiling by multiplex assays

• Histopathological assessment:

  • Tissue sections stained with hematoxylin and eosin for cellular infiltration

  • Immunohistochemistry for immune cell markers

  • Special stains for microglial activation and astrogliosis

• Functional assessments:

  • Behavioral testing for neurological deficits

  • Electrophysiological recordings

  • Blood-brain barrier permeability assays

• Molecular analyses:

  • Gene expression profiling of inflammatory mediators

  • Western blotting for signaling pathway activation

  • Flow cytometry of isolated CNS inflammatory cells

This severity classification is based on experimental CNS infection models and may require adjustment for specific experimental systems .

How can researchers verify the biological activity of recombinant rabbit TNF preparations?

Verification of TNF biological activity is crucial before experimental use:

• Cytotoxicity assays: Use sensitive cell lines like L929 fibroblasts or WEHI-164 cells in the presence of actinomycin D. The ED50 for mouse TNF-alpha typically ranges from 8-50 pg/mL .

• NF-κB reporter assays: Cells transfected with an NF-κB responsive element driving luciferase or other reporter genes.

• Phospho-protein detection: Western blotting for phosphorylated signaling molecules downstream of TNF receptor activation (p38 MAPK, JNK, IκB).

• Cell surface marker induction: Flow cytometric analysis of TNF-induced adhesion molecules on endothelial cells.

• Pilot in vivo testing: Small-scale tests measuring expected biological responses (e.g., CSF leukocytosis after intrathecal administration) .

What are common pitfalls when working with recombinant rabbit TNF in research applications?

Researchers should be aware of several potential pitfalls:

• Loss of activity: TNF activity can diminish due to improper reconstitution, storage, or excessive freeze-thaw cycles.

• Endotoxin contamination: Bacterial endotoxins in recombinant protein preparations can cause inflammatory responses that confound experimental results.

• Batch-to-batch variability: Differences in specific activity between production lots can affect experimental reproducibility.

• Species-specificity issues: Although cross-species reactivity exists, potency may vary across species. Murine TNF is active in rabbits but may have different potency compared to rabbit TNF .

• Carrier protein interference: BSA or other carrier proteins in TNF preparations may interfere with certain applications, necessitating carrier-free formulations .

• Short half-life in vivo: TNF has a relatively short circulatory half-life, requiring consideration in dosing schedules for in vivo experiments.

How can recombinant rabbit TNF be utilized in models of infectious disease pathogenesis?

Recombinant TNF serves as a valuable tool in infectious disease research:

• Experimental meningitis models: TNF has been identified as a key determinant in tuberculous meningitis pathogenesis. In rabbit models, CSF levels of TNF-α correlate with disease severity, including leukocytosis, protein accumulation, and meningeal inflammation .

• Comparative virulence studies: Different microbial strains induce varying levels of TNF, correlating with pathogenicity. For example, M. bovis Ravenel induces higher TNF levels than BCG strains, corresponding with greater pathology .

• Mechanistic investigations: Recombinant TNF can help dissect pathways of infection-induced inflammation. For example, recombinant BCG Montreal expressing murine TNF-α showed enhanced virulence compared to vector control, demonstrating TNF's causal role in pathogenesis .

• Therapeutic targeting studies: Models using recombinant TNF can assess the efficacy of TNF-modulating therapies. For instance, thalidomide therapy protected rabbits with experimental tuberculous meningitis by inhibiting TNF-α production .

What novel approaches are being developed to enhance stability and delivery of recombinant TNF for research applications?

Emerging approaches include:

• PEGylation: Addition of polyethylene glycol moieties to increase half-life and reduce immunogenicity.

• Fusion proteins: Creation of TNF fusion constructs with antibody fragments or other proteins for targeted delivery or enhanced stability.

• Nanoparticle formulations: Encapsulation in biodegradable nanoparticles for controlled release and tissue-specific targeting.

• Gene delivery approaches: Viral vectors or non-viral delivery systems for localized TNF expression in specific tissues.

• Site-specific modifications: Engineering TNF proteins with modified receptor binding profiles or signaling capabilities.

These approaches have potential applications in both basic research and therapeutic development, allowing more precise control over TNF activity in experimental systems.