Construction of Lentiviral Expression Vector Containing Extracellular Domain of Human Hepatocyte Growth Factor Receptor and Its Expression in 293T Cell / 生物医学工程学杂志

ABSTRACT

This research aims to construct a lentiviral expression vector carrying the extracelluar domain (ED) of human hepatocyte growth factor receptor (C-Met), and to express it in transfected 293T cells. The extracellular domain of C-Met was amplified by RT-PCR, ligated with lentiviral expression vector p RRL-CMV-ED, and then expressed in 293T cell line. The expressed protein was purified and identified by RT-PCR and Western blot. The enzyme digestion and sequence analysis showed that the lentiviral expression vector p RRL-CMV-ED was constructed correctly. The size of amplified genes was about 2 700 bp. The purified protein with Ni-affinity column was about 105 kD analyzed by SDS-PAGE. The Western blot and ELISA results showed that the expressed protein which could bind to HGF specifically was the extracelluar domain of human hepatocyte growth factor receptor. This research may lay a foundation for further study of anti-C-MET monoclonal antibody and neutralizing antibody.