Difference between revisions of "Part:BBa K2568006"
 
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<partinfo>BBa_K2568006 short</partinfo>
 
<partinfo>BBa_K2568006 short</partinfo>
  
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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.  
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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.
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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.
  
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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.
 
  
  
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'''Material Preparation'''
 
'''Material Preparation'''
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*MDBK cells (2×106 cells·mL-1)  
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*MDBK cells (2×106 cells/mL)  
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*plasmid (20 μg) with concentration greater than 1 μg·μL-1 for each sample
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*plasmid (20 μg) with concentration greater than 1 μg/μL for each sample
 
   
 
   
 
'''Transfection'''
 
'''Transfection'''
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*Wash and resuspend the cells with pre-chilled PBS after trypsinization.  
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*Wash and resuspend cells with pre-chilled PBS after trypsinization.  
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*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.
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*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.  
 
*Place the solution in a pre-chilled shock cup (2 mm) which is sterilized with ethanol in ice bath for 1 min.  
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*Give the shock cup shocks for three times with the cell electroporation apparatus (350 V, 500μs) and each interval is 1min.  
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*Give the shock cup shocks for three times with the cell electroporation apparatus (350 V, 500μs) and each interval is 1 min.  
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*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.
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*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.
 
*Remove the supernatant from the 6-well plate and replace it with fresh medium.
  
 
'''Test'''
 
'''Test'''
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*Observe GFP expression in the cells using fluorescence microscopy.
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*Observe GFP expression in cells using fluorescence microscopy.
  
  
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===Characterization===
 
===Characterization===
  
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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.
+
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'''
 
'''Nucleic acid gel electrophoresis'''
  
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After constructing the plasmid with Nectin 4, we conducted the nucleic acid gel electrophoresis and DNA sequencing to ensure the accuracy.
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After constructing the plasmid carrying Nectin 4, we conducted the nucleic acid gel electrophoresis and DNA sequencing to validate its accuracy.  
  
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[[Image:T--Jiangnan--Nucleic_acid_gel_electrophoresis_.jpg|center|thumb|200px|Figure 1. '''Nucleic Acid Gel Electrophoresis''']]
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[[Image:T--Jiangnan--Nucleic_acid_gel_electrophoresis_.jpg|center|thumb|200px|'''Figure 1.Nucleic Acid Gel Electrophoresis'''
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 +
M: DL10000 DNA Marker; 1: pcDNA-DogN4-FLAG.
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The 5400 bp band: pcDNA3.1(+) plasmid backbone; the 1570 bp band: Nectin 4 fragment.
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]]
  
 
'''RT-PCR'''
 
'''RT-PCR'''
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We detected Nectin 4 expression at the transcriptional level in steady transplanted cell strains with RT-PCR.
 
We detected Nectin 4 expression at the transcriptional level in steady transplanted cell strains with RT-PCR.
  
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[[Image:T--Jiangnan--RT_PCR.png|center|thumb|500x800px|Figure 2. '''The expression of Nectin 4 mRNA was detected in MDBK-N4 cells by RT-PCR''']]
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[[Image:T--Jiangnan--RT_PCR.png|center|thumb|250px|'''Figure 2. Nectin 4 mRNA expression detected in MDBK-N4 cells by RT-PCR.'''
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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.
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The 153 bp band: GADPH mRNA; the 1570 bp band: Nectin 4 mRNA.
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]]
  
 
'''Western Blot'''
 
'''Western Blot'''
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We detected Nectin 4 expression at the translational level in steady transplanted cell strains with Western blot.
 
We detected Nectin 4 expression at the translational level in steady transplanted cell strains with Western blot.
  
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[[Image:T--Jiangnan--western_blot.jpg|center‎|thumb|300px|Figure 3. '''The expression of Nectin 4 was detected in MDBK-N4 cells by Western blot''']]
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 +
[[Image:T--Jiangnan--western_blot.jpg|center|thumb|350px|'''Figure 3. Nectin 4 expression detected in MDBK-N4 cells by Western blot.'''
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56kDa: Nectin 4 protein molecular weight.  36kDa: GAPDH protein molecular weight.
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MDBK-N4 cells showed a clear band at 56 kDa, indicating that MDBK-N4 cells can stably express Nectin 4 protein.
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]]
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'''Indirect Immunofluorescence'''
 
'''Indirect Immunofluorescence'''
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<div>
 
<div>
 
     <ul>  
 
     <ul>  
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                 <li style="display: inline-block;"> [[Image:T--Jiangnan--immunofluorescence_40.jpg|thumb|300px|Figure 3. '''Expression of Nectin4 was detected in MDBK-N4 cells by immunofluorescence (×40)''']]</li>                                  
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                 <li style="display: inline-block;"> [[Image:T--Jiangnan--immunofluorescence_40.jpg|thumb|380px|'''Figure 4. Nectin 4 expression detected in MDBK-N4 cells by immunofluorescence (×40). '''
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                 <li style="display: inline-block;"> [[Image:T--Jiangnan--immunofluorescence_100.jpg|thumb|300px|Figure 4. '''Expression of Nectin4 was detected in MDBK-N4 cells by immunofluorescence (×100)''']]</li>
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A: MDBK cells; B: MDBK-N4 cells.
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Green and blue each represents Nectin 4 and nuclei of cells.]]</li>                                                                                              
 +
                 <li style="display: inline-block;"> [[Image:T--Jiangnan--immunofluorescence_100.jpg|thumb|380px|'''Figure 5. Nectin 4 expression detected in MDBK-N4 cells by immunofluorescence (×100).'''
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A: MDBK cells; B: MDBK-N4 cells.
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Green and blue each represents Nectin 4 and nuclei of cells.]]</li>
 
     </ul>
 
     </ul>
 
</div>
 
</div>
  
  
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[[Image:T--Jiangnan--Envelope_protein_of_CDV.jpg|center|thumb|500x800px|Figure 5. '''Envelope protein of Canine Distemper Virus was detected in MDBK-N4 cells by fluorescent microscope (×40)''']]
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[[Image:T--Jiangnan--Envelope_protein_of_CDV.jpg|center|thumb|500x800px|'''Figure 6. Envelope protein of CDV detected in MDBK-N4 cells by fluorescent microscope (×40).'''
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A: MDBK cells; B: MDBK-N4 cells.
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Green and blue each represents viral envelope protein and nuclei of cells.]]
  
  

Latest revision as of 06:25, 29 September 2018


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

  • 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.
  • 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