Construction of a eukaryotic expression vector of fibroblast activation protein and establishment of its stable over-expression in the oral squamous cell carcinoma

Abstract

This study aimed at constructing fibroblast activation protein (FAP) over-expression lentivinus vectors to investigate transfection in SCC9 cell lines and establish a stable FAP over-expression oral squamous cell line. The cDNA of FAP gene from an oral squamous cell carcinoma (OSCC) tissue was amplified by polymerase chain reaction (PCR) and subcloned into eukaryotic expression vector pCDH-CMV-MCS-EF1-copGFP. The recombinant plasmid was sequenced and then transfected into an SCC9 cell line. Subsequently, the SCC9 cell line that over-expressed FAP stably was established by fluorescence activated cell sorting (FACS). The expression of green fluorescent protein (GFP) was detected with fluorescence microscopy, and the over-expression of FAP was identified by real-time PCR and Western blot. The FAP gene was amplified by PCR and then cloned into the vector, whose sequence was identical to that in the GenBank. GFP was expressed in the transfected cells. Furthermore, FAP over-expression in the transfected cells was detected by real-time PCR and Western blot. The recombinant eukaryotic expression vector pCDH-FAP was constructed successfully. This result provides a foundation for further studies on the function of FAP in vitro.