Part:BBa_K2568006

Virus receptor Nectin 4 with Kozak sequence and FLAG-tag

The way how viruses enter into cells is mainly mediated by host receptors. We have established an association between virus Baltimore subtyping and host receptors, and found 4 internalization receptors and 2 attachment molecules that are necessary to mediate the entry of Baltimore types I to V. Our chassis cell MDBK has most internalization receptors and adhesion molecules, but lacks Nectin 4 and TfR to enable the production of most of the commonly seen animal viruses.

Nectin 4 is one of the receptors that mediates the entry of canine distemper virus (CDV). As a kind of cattle cell, MDBK cannot be naturally infected by canine viruses, but can become sensitive to CDV infection if Nectin 4 was functionally expressed on cell surface. Therefore, we are motivated to construct the Nectin 4 biobrick to achieve the goal of cultivating multiple viruses in MDBK cells.

Usage and Biology

Material Preparation

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

Transfection

• Wash and resuspend cells with pre-chilled PBS after trypsinization.
• After centrifugalization for 5 min, add PBS (200 μl) to resuspend cells(2×106cells/mL) 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 1 min.
• Wash the shock cup with DMEM medium containing 10% FBS and transfer cells to a 6-well plate for a final volume of 2 mL/well.
• Remove the supernatant from the 6-well plate and replace it with fresh medium.

Test

• Observe GFP expression in 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 this protein.

Nucleic acid gel electrophoresis

After constructing the plasmid carrying Nectin 4, we conducted the nucleic acid gel electrophoresis and DNA sequencing to validate its accuracy.

Figure 1.Nucleic Acid Gel Electrophoresis M: DL10000 DNA Marker; 1: pcDNA-DogN4-FLAG. The 5400 bp band: pcDNA3.1(+) plasmid backbone; the 1570 bp band: Nectin 4 fragment.

RT-PCR

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

Figure 2. Nectin 4 mRNA expression detected in MDBK-N4 cells by RT-PCR. M: DL500 DNA Marker; 1-2: MDBK cells and MDBK-N4 cells for Nectin 4;3-4: MDBK cells and MDBK-N4 cells for GAPDH. The 153 bp band: GADPH mRNA; the 1570 bp band: Nectin 4 mRNA.

Western Blot

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

Figure 3. Nectin 4 expression detected in MDBK-N4 cells by Western blot. 56kDa: Nectin 4 protein molecular weight. 36kDa: GAPDH protein molecular weight. MDBK-N4 cells showed a clear band at 56 kDa, indicating that MDBK-N4 cells can stably express Nectin 4 protein.

Indirect Immunofluorescence

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

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  Figure 4. Nectin 4 expression detected in MDBK-N4 cells by immunofluorescence (×40). A: MDBK cells; B: MDBK-N4 cells. Green and blue each represents Nectin 4 and nuclei of cells.
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  Figure 5. Nectin 4 expression detected in MDBK-N4 cells by immunofluorescence (×100). A: MDBK cells; B: MDBK-N4 cells. Green and blue each represents Nectin 4 and nuclei of cells.

Figure 6. Envelope protein of CDV detected in MDBK-N4 cells by fluorescent microscope (×40). A: MDBK cells; B: MDBK-N4 cells. Green and blue each represents viral envelope protein and nuclei of cells.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 220
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1041
  Illegal BsaI.rc site found at 1369
  Illegal SapI.rc site found at 1168