Difference between revisions of "Part:BBa K1607010"

Revision as of 06:45, 24 August 2015

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. 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

To get this coding sequence, we found the AA sequence of this SCFV on NCBI first (PDB: 3H3B_B). The 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.see oligos and protocols

The 601bp sequence synthesized by LCR assembly actually included an EcoRI site and an XbaI site at the ends of it. These two restriction sites were designed for pPICZα-a yeast expression vector, but the yeast expression plan was eventually given up due to its complexity. After the LCR assembly, standard Biobrick prefix and suffix were added to the sequence by PCR, replacing the originally designed EcoRI and XbaI. Finally, the standard part was cloned to PSB1C3 vector.