Part:BBa_K4961001

PEE6.4

The PEE6.4 vector is a Glutamine Synthetase (GS) gene expression system vector from PLonza Biotech. The target gene can be expressed at a high level in mammalian cells by using glutamine synthase to screen markers. This is a commercial carrier, and the project designer's laboratory has made some improvements for subsequent experiments.

PEE6.4 BBa_K4961001

Profile

• Name: PEE6.4
• Base Pairs: 5067 bp
• Origin: Commercial plasmids produced by biological companies
• Properties: GS System Mammalian Cell Protein Expression Vector, Antibody Expression Vector.
• Notes: The plasmid is a commercially available vector, which was subjected to a series of modifications by the designer's laboratory after its pre-introduction.

Construt Design

pEE6.4 vector as a plasmid backbone, used for constructing recombinant plasmids with light chain DNA fragment of the antibody.

Experimental Procedure

In order to construct our complete antibody expression plasmid, we asked Gene Synthesis company to synthesise the DNA fragments. Then, using the technique of molecular cloning, we inserted the heavy chain DNA fragment of the antibody into the pEE12.4 vector and the light chain DNA fragment of the antibody into the pEE6.4 vector. We transformed the expression plasmid into E. coli competent cells, and then coated them on LB solid medium plates containing ampicillin and cultured them overnight at 37℃.

Figure 1. Purity verification

Characterization/Measurement

Now we have the product we want to develop, the 33D9 antibody, which binds specifically to paclitaxel. With the double antibody sandwich method (Figure 6) we can detect the concentration of ADC coupled to ptx in different media. Before applying it to a real scenario, we have to make a standard curve for our product to determine its upper and lower limits of detection. The results of the double-antibody sandwich method showed that we successfully made a standard curve that could detect 52B8hG1ptx. By analogy, any drug coupled to paclitaxel, which itself can be directly coupled to fluorescence or indirectly coupled to fluorescence, can be detected in different solutions using our antibody. This provides strong experimental data to support the development of our ELISA kit.

Figure 2. ELISA results: Standard Curve of 52B8G1PTX

Reference:

https://www.novopro.cn/vector/Vgezdmobr

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal XbaI site found at 2523
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BglII site found at 6975
  Illegal XhoI site found at 1790
  Illegal XhoI site found at 2505
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal XbaI site found at 2523
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal XbaI site found at 2523
  Illegal NgoMIV site found at 2955
  Illegal NgoMIV site found at 4296
  Illegal NgoMIV site found at 4579
  Illegal AgeI site found at 795
  Illegal AgeI site found at 2493
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI.rc site found at 759
  Illegal BsaI.rc site found at 6107
  Illegal SapI site found at 5024
  Illegal SapI.rc site found at 2179
  Illegal SapI.rc site found at 4145
  Illegal SapI.rc site found at 4355