Construction of eukaryotic expression vector of major histocompatibility complex class I -related chain A and establishment of its stable transfected Tca8113-Tb cell line

To construct the eukaryotic expression vector, encoding major histocompatibility complex class I-related chain A gene (MICA), for the further research of transfecting Tca8113-Tb cell line(a metastatic cell line of brain metastasis from human tongue cancer Tca8113 cells in nude mouse), and to establish a stable MICA overexpression oral squamous cell line. cDNA of MICA gene from pCMV-SPORT6-MICA was amplified by PCR, and subcloned into eukaryotic expression vector pEGFP-N1 marked with green fluorescent protein (GFP). The recombinant plasmid was sequenced and transfected into Tca8113-Tb cell line by lipofectamine 2000. After screen culture by G418, stable tranfected Tca8113-Tb cell line was established using definite dilution method. The expressions of GFP protein was viewed directly with fluorescence microscopy and the overexpression of MICA was identified by RT-PCR, real time PCR and immunocytochemistry. The MICA gene was amplified by PCR and then cloned into the vector, whose sequence was identical to that in