Difference between revisions of "Part:BBa K2568007"

Latest revision as of 06:28, 29 September 2018

Virus receptor TfR with Kozak sequence and His-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.

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

Characterization

Nucleic acid gel electrophoresis

After constructing the plasmid with TfR, we conducted the nucleic acid gel electrophoresis and DNA sequencing to ensure the accuracy.

Figure 1. Nucleic Acid Gel Electrophoresis M: DL10000 DNA Marker; 1: PLVX-TfR. The 8787 bp band: PLVX plasmid backbone; the 1570 bp band: TfR fragment.

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 AgeI site found at 721
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2568007 short</partinfo>
-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.
+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.
+TfR is one of the receptors that mediates the entry of canine parvovirus (CPV). As a kind of cattle cell, MDBK cannot be naturally infected by canine viruses, but can become sensitive to CPV infection if TfR was functionally expressed on cell surface. Therefore, we are motivated to construct the TfR biobrick to achieve the goal of cultivating multiple viruses in MDBK cells..
-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. TfR protein is one of the receptors of canine parvovirus (CPV), and it can strongly bind to CPV. As a kind of cattle cell, MDBK can not be infected by canine virus. But if MDBK can express TfR protein on the cell membrane, it can be infected by CPV. To achieve the goal of culturing mutiple viruses in the MDBK cell strain, we constructed TfR biobrick which can be expressed to Nectin 4 protein and transfected it into MDBK cells. We hopes that TfR protein can be successfully expressed on the membrane, the CDV can bind to it and invade MDBK cells and then multiply.
@@ Line 15: / Line 16: @@
 After constructing the plasmid with TfR, we conducted the nucleic acid gel electrophoresis and DNA sequencing to ensure the accuracy.
-[[Image:T--Jiangnan--Nucleic_acid_gel_electrophoresis_TfR.jpg|center|thumb|200px|Figure 1. '''Nucleic Acid Gel Electrophoresis ''']]
+[[Image:T--Jiangnan--Nucleic_acid_gel_electrophoresis_TfR.jpg|center|thumb|200px|'''Figure 1. Nucleic Acid Gel Electrophoresis '''M: DL10000 DNA Marker; 1: PLVX-TfR.
+The 8787 bp band: PLVX plasmid backbone; the 1570 bp band: TfR fragment.
+]]