Part:BBa_K1607011
Part Description
The anti-p185her2/neu antibody chA21 mediates specific inhibitory effects on p185her2/neu-overexpressed cancer cells, as well as human breast and ovarian cancer xenograft [1] This part is the coding sequence of chA21 SCFV, fused with an EGFP. The SCFV should be able to specifically bind to tumor antigen p185her2/neu-ECD (“ECD” stands for “extracellular domain”). [1] Zhou H, Zha Z, Liu Y, et al. Structural Insights into the Down-regulation of Overexpressed p185her2/neu Protein of Transformed Cells by the Antibody chA21[J]. Journal of Biological Chemistry, 2011, 286(36): 31676-31683.
The obtainment of DNA sequence
The obtainment of DNA sequence: To get this coding sequence of the SCFV, we found the AA sequence of it on NCBI first (PDB: 3H3B_B). The SCFV AA sequence was then reverse translated into DNA sequence. The DNA sequence was analyzed by gene2oligo and 36 oligos were given as a result. We synthesized the 36 oligos and obtained the 601bp fragment of SCFV coding sequence by LCR assembly using these oligos. The SCFV sequence was then connected to the EGFP sequence by SOE PCR, a (Gly4Ser)3 linker was inserted.
This part is designed for FRET analysis. An Mcherry will be fused with the antigen, and by measuring the FRET efficiency we can detect the antibody-antigen interaction. Oligos:
| None |