To P185 And ~ (erbb-2) Protein-targeting Single-chain Antibody Screening

erbB-2,or HER2/neu,is one of cancer genes belong to tyrosineproteinkinase family, which coding a transmembrane phosphaprotein( M.W. 185 kD). In about 25% of breast cancer,ovary cancer and such cancer,overexpression or exceptional activation of HER2/neu have been founded. For the sake of getting anti-P185^erbB2 ScFv,this rearch carry through the nether work:At first,imposed bioinformatics technology and reported production,a domain approached to transmembrane of P185^erbB2 was cognized as a potential anti-tumor target and the anticipant artificial antigen. Total RNA was distilled from cell line of breast cancer - sk-br-3 and a segment( aa 505-625 ) was enlarged and cloned into expression vector pET-22b(+). The expression of such gene segment was in high efficiency (approximate 30% of total dissoluble protein) and purified protein was get by means of HiTrap chelating column.In the next,a phage display ScFv library in high capacity was panning three cycles used the above artificial antigen and several positive clone was analysed by means of ELISA in the aspect of specific and comparative appetency at protein level.At last,two good clone was pick out and corresponding ScFv genes was clone into vector pTTG-e witch is a expression vector with e-tag. This work make the basis of more analyse of specific and comparative appetency at cell level and organization level.