Fibronectin (FN), a multifunctional glycoprotein naturally present in the extracellular matrix and plasma, is an ideal functional molecule for viral vector surface engineering. FN possesses unique advantages: its RGD cell-binding domain can specifically recognize integrin receptors to achieve cell targeting; the human-derived sequence ensures biocompatibility and reduces immunogenicity; and it exhibits excellent compatibility with various viruses (retrovirus, lentivirus, adenovirus, adeno-associated virus).
Structural and Functional Domains
|
Functional Domain |
Position |
Molecular Characteristics |
Application in Viral Vector Engineering |
|
RGD Cell-Binding Domain |
FN Ⅲ-10 (aa1493–1495) |
Arg-Gly-Asp sequence |
Targets integrin-highly expressing cells and enhances viral adsorption |
|
Synergy Domain (PHSRN) |
FN Ⅲ-9 (aa1370–1374) |
Pro-His-Ser-Arg-Asn sequence |
Enhances RGD activity and improves virus-cell binding affinity |
|
Heparin-Binding Domain (HepII) |
FN Ⅲ-12–14 (aa1541–1810) |
Basic amino acid-enriched region |
Binds to viral surfaces or assists virus interaction with cellular sulfated polysaccharides |
|
CS-1 Site |
Type Ⅲ CS alternatively spliced domain (aa1811–1840) |
Binds to α4β1 integrin |
Targets hematopoietic stem cells |
Mechanism of Action
Cell surface VLA-4 and VLA-5 bind to the CS-1 site and C-domain of fibronectin, respectively, while retroviral vectors can bind to the H-domain. This technology markedly improves transduction efficiency, reduces viral dosage by 50–80%, and minimizes cellular toxicity.
Co-coating with anti-CD3 antibody (OKT3) and FN synergistically activates T cells and enhances transduction; spinoculation (800–1000×g) further boosts efficiency; and multiple transduction strategies increase the proportion of high-expression cells.
Usage Protocol
- Coated Plate Preparation：Thaw the FN stock solution (1 mg/mL) on ice, dilute to 25–50 μg/mL with sterile PBS; add to non-tissue culture-treated plates at 2 mL per well (6-well plate); incubate at room temperature for 2 hours or at 4°C overnight; block with 2% BSA/PBS for 30 minutes; wash once with PBS, then use immediately or store at 4°C (use within 48 hours).
- Virus Pre-Binding (Optional)：Add lentiviral supernatant to the coated wells and incubate at 32°C for 1–2 hours to allow virus-FN binding.
- Cell Transduction：Harvest target cells (e.g., activated T cells, 0.5–1×10⁶ cells/mL) and seed into coated wells; add virus at an MOI of 1–10; perform spinoculation at 800–1000×g, 32°C for 1–2 hours; continue incubation at 37°C with 5% CO₂; assess transduction efficiency 24–48 hours later.
- Key Parameter Optimization：FN concentration: 25–50 μg/mL (optimized according to cell type); viral MOI: 1–10 (RetroNectin significantly reduces required MOI); cell density: 0.5–1×10⁶/mL (avoid over-confluence).
Product Recommendation
YEASEN offers HiActi® Recombinant Human Fibronectin Protein, produced via mammalian cell expression, isolation, purification, and filter sterilization. This product features high purity, high bioactivity, low endotoxin level, and excellent stability.
HiActi™ Recombinant Human Fibronectin Product
High Purity
Figure 1. Human Fibronectin analyzed by SDS-PAGE under reducing conditions. Purity is greater than 90%.
High Bioactivity
Figure 2. The ED50 as determined by a celI proliferation assay using B16-F1 mouse melanoma cells is less than 1.1 μg/mL, corresponding to a specific activity of > 1.17 X 107 IU/mg. Fully biologically active when compared to standard.
Product Ordering Information
|
Cat. No. |
Product Name |
Size |
Expression System |
|
92619ES |
1 mg / 5 mg / 10 g / 100 mg |
CHO |