Fusion of EGFP and porcine α 1,3GT genes decrease GFP expression

Abstract

Objective To investigate the effect of fusion proteins expressed by the fused gene of porcine α 1, 3 galactosyltransferase (α1, 3 GT) and enhanced green fluorescent protein (EGFP) on the green fluorescence intensity of EGFP. Methods The fragment containing α 1, 3GT was firstly recovered after the pcDNA3.1- α 1, 3GT recombinant vector were digested with Bam HI and Eco RI, and then, the resultant fragment was ligated to the pEGFP-N1 vector which was also digested with the same enzymes. The new recombinant eukaryotic expression pEGFP/α 1, 3GT vector was obtained and sequenced. The pEGFP/α 1, 3GT was used to transfect human lung carcinoma cells A549 and HEKC 293FT, and the expression of EGFP was quantitatively analyzed by fluorescent microscope and flow cytometry. Results The positive percentage of A549 was 80.5%, and that of 293 FT was 86.5% 48 hours after the two cell lines both were transfected by pEGFP-N1. The positive percentage of A549 was 75.8%, and that of 293 FT was 81.2% 48 hours after the two cell lines were transfected by pEGFP/α 1, 3GT. The mean fluorescence intensities of A549 transfected with pEGFP-N1 and pEGFP/α 1, 3GT were 1.21 and 0.956, respectively when compared with that of A549 without transfection. Meanwhile, the those of the 293FT that were transfected with pEGFP-N1 and pEGFP/α 1, 3GT were 7.66 and 1.00, respectively when compared with that of 293FT cells without transfection. Conclusions These results suggested that the expression of EGFP gene fused with porcine α 1, 3GT gene was partly inhibited.