The Study of the Targeting Selectivity and Binding the Surface of Breast Cancer Cells with the Fusion Protein Anti-HER2-ScFv-GFP in vitro Experiments

Abstract: The goal of this study was to test the targeting binding efficiency of the fusion protein Anti- HER2-ScFv-GFP on the surface of breast cancer cells. We constructed the eukaryotic expression system pFAST Bac to Bac HT A/Anti-HER2-ScFv-GFP/Tn-5B1-4 and the prokaryotic system pBAD His B/Anti-HER2-ScFv- GFP/TOP10. And then the fusion protein Anti-HER2-ScFv-GFP was separated to get the purification with Ni2+- NTA argrose from the eukaryotic expression system pFAST Bac to Bac HT A/Tn-5B1-4 and the prokaryotic expression system pBAD His B/TOP10. Then we incubated SKBR3 (HER2+ cell) and MCF7(HER2- cell) containing the purification of the fusion proteins in 24 h， eluted these cells with 1×PBS three times， examined the targeting binding efficiency of the fusion protein Anti-HER2-ScFv-GFP on the surface of breast cancer cells with laser confocal microscopy system. Consequently， apparent green fluorescence was detected in SKBR3 cells. Fusion proteins from eukaryotic expression system showed a higher binding efficiency than those from prokaryotic expression system. Incubation with high concentration fusion proteins induced shrinking in SKBR3 cell. In contrast， fusion proteins were readily eluted from the HER2 negative cell MCF7， without obvious fluorescence detected. The standard GFP was readily eluted from the HER2 positive cell SKBR3， too. Fusion protein (Anti-HER2-ScFv-GFP) from these two systems can all bind to the surface of SKBR3 cell， but proteins from eukaryotic system showed a higher binding capacity than those from prokaryotic system. This suggested that GFP can report the developing of the breast cancer cells SKBR3 with anti HER2 ScFv and engineer antibodies selected to co-target critical functional pairs of HER2 on the surface of SKBR3 in vitro.

CSTR: 32200.14.cjcb.2011.05.0005