Recombinant Merkel Cell Polyomavirus (MCPyV) Virion Preparation: A Technique to Produce High-titer Recombinant MCPyV Virion
Published: April 30, 2023
Abstract
Source: Wei Liu et al. Merkel Cell Polyomavirus Infection and Detection, J. Vis. Exp. (2019)
In this video, we show the preparation of Merkel cell polyomavirus or MCPyV virions using 273TT cells. This technology helps to produce high-titer of the recombinant MCPyV virions.
Protocol
1. Recombinant MCPyV virion preparation Digest 50 µg of pR17b plasmid (carrying MCPyV genome) with 250 U of BamHI-HF in a 200 µL volume (4 h at 37 °C) to separate the viral genome from the vector backbone (Figure 1). Add 1200 µL of buffer PB (supplemented with 10 µL of 3 M NaAc, pH 5.2) to the digested DNA and purify over 2 miniprep spin columns (20 µg DNA capacity). Elute the digested pR17b plasmid from each column with 200 µL of …
Representative Results
Figure 1. Production of MCPyV virion using recombinant viral genome. (A) A plasmid map of pR17b (MCPyV genome plasmid). (B) A representative picture of an MCPyV virion sample harvested and purified over a gradient. Arrow marks the band of MCPyV virions concentrated in the core of the gradient. (C) The viral genome copy number in each gradien…
Divulgaciones
The authors have nothing to disclose.
Materials
| DMEM/F12 medium | Thermo Fisher Scientific | 11330-032 | |
| Recombinant Human EGF Protein, CF | R&D systems | 236-EG-200 | Store at -80 degree celsius |
| CHIR99021 | Cayman Chemical | 13122 | Store at -80 degree celsius |
| CHIR99021 | Sigma | SML1046 | Store at -80 degree celsius |
| Collagenase type IV | Thermo Fisher Scientific | 17104019 | |
| Dispase II | Roche | 4942078001 | |
| Antibiotic-Antimycotic | Thermo Fisher Scientific | 15240-062 | Protect from light |
| DMEM medium | Thermo Fisher Scientific | 11965084 | |
| Paraformaldehyde | Sigma | P6148 | |
| Anti-MCPyV LT (CM2B4) | Santa Cruz | sc-136172 | Lot # B2717 |
| MCV VP1 rabbit | Rabbit polyclonal serum #10965 | https://home.ccr.cancer.gov/lco/BuckLabAntibodies.htm |
Citar este artículo
Recombinant Merkel Cell Polyomavirus (MCPyV) Virion Preparation: A Technique to Produce High-titer Recombinant MCPyV Virion. J. Vis. Exp. (Pending Publication), e20358, doi: (2023).