Difference between revisions of "Part:BBa K2568006"

Revision as of 13:43, 27 September 2018

Virus receptor Nectin 4 with Kozak sequence and FLAG-tag

The way in which viruses enter into cells is mainly mediated by the host receptor. If a cell can simultaneously express receptors like Nectin 4 protein and TfR protein, and adhesion molecules like heparan sulfate, this cell can be infected by most viruses and is expected to solve the cultivation problem of most viruses.

Our chassis cell MDBK, which is a commonly used cell line in the production of bovine vaccines, has most of the essential receptors and adhesion molecules, and only lacks Nectin 4 protein and TfR protein receptors. Nectin 4 protein is one of the receptors of canine distemper virus (CDV), and its lgV structure forms the strongest binding ability of all receptors with the H protein in CDV. As a kind of cattle cell, MDBK can not be infected by canine virus. But if MDBK can express Nectin 4 protein on the cell membrane, it can be infected by CDV. To achieve the goal of culturing mutiple viruses in the MDBK cell strain, we constructed Nectin 4 biobrick which can be expressed to Nectin 4 protein and transfected it into MDBK cells. When Nectin 4 protein was successfully expressed on the membrane, the CDV can bind to it and invade MDBK cells and then multiply.

Usage and Biology

Material Preparation

• MDBK cells (2×106 cells·mL-1)
• plasmid (20 μg) with concentration greater than 1 μg·μL-1 for each sample

Transfection

• Wash and resuspend the cells with pre-chilled PBS after trypsinization.
• After centrifugalization for 5 min, add PBS (200 μl) to resuspend cells(2×106cells·mL-1) at room temperature, and then add plasmid(20 μg), salmon sperm DNA(10 μg) together.
• Place the solution in a pre-chilled shock cup (2 mm) which is sterilized with ethanol in ice bath for 1 min.
• Give the shock cup shocks for three times with the cell electroporation apparatus (350 V, 500μs) and each interval is 1min.
• Wash the shock cup with DMEM medium containing 10% FBS and transfer the cells to a 6-well plate for a final volume of 2 mL·well-1.
• Remove the supernatant from the 6-well plate and replace it with fresh medium.

Test

• Observe GFP expression in the cells using fluorescence microscopy.

Characterization

After transfecting the Nectin 4 biobrick into MDBK cells, we conducted a series of experiments which include mRNA, protein and cells level to verify the function of the protein.

Nucleic acid gel electrophoresis

After constructing the plasmid with Nectin 4, we conducted the nucleic acid gel electrophoresis and DNA sequencing to ensure the accuracy.  <img src="https://static.igem.org/mediawiki/2018/7/78/T--Jiangnan--Nucleic_acid_gel_electrophoresis.png?tdsourcetag=s_pctim_aiomsg" alt="some_text">

Figure 1. Cells grown in GFP-medium containing the arabinose promoter regulating GFP expression. Induced at time=0h.

RT-PCR

We detected Nectin 4 expression at the transcriptional level in steady transplanted cell strains with RT-PCR.

Western Blot

We detected Nectin 4 expression at the translational level in steady transplanted cell strains with Western blot.

Indirect Immunofluorescence

We detected Nectin 4 and viral coat protein expression by Indirect Immunofluorescence

Sequence and Features