Construction Of Eukaryotic Expression Vector Of Sika Deer PDGFA Gene And Expression In 293T Cells

Platelet-derived growth factor A(PDGFA),a member of a large family of platelet-derived growth factors,is produced by a variety of cells.It activates the PDGF/PDGFR pathway by binding to and activating the highly specific cell surface receptor PDGFRa,thus exerting corresponding biological effects.Present studies have shown that PDGFA plays an important role in the proliferation and migration of various cancer cells.The rapid growth and regeneration of velvet antler are unique in nature,and it has become an ideal model for regenerative biology research.Its rapid proliferation research may also become a new entry point for cancer treatment.In this study,based on the gene sequence of cattle in Genbank,homologous primers of the PDGFA gene which are added enzyme digestion sites with Hind ?and BamH ? was designed.The cDNA sequence of PDGFA gene from sika deer antler tissue was obtained by RT-PCR.The sequence analysis showed that the PDGFA gene was 663bp,encoding 196 amino acids.By comparison,it was found that the PDGFA protein of sika deer had high homology with white-tailed deer,cattle and goats,98%,97%and96%respectively,which proved that the PDGFA gene of sika deer was successfully cloned.The eukaryotic expression vector of pEGFP-C1-PDGFA was constructed.Eukaryotic expression vectors pEGFP-C1-PDGFA and empty vectors pEGFP-C1 were transfected into 293T cells instantaneously.A large amount of green fluorescence was observed under fluorescence microscope and the transfection efficiency is 60-70%which proved that the transfection was successful.The relative expression level of PDGFA gene in pEGFP-C1-PDGFA transfected cells was about 280 times higher than that in the control group,which indicated that PDGFA gene was successfully expressed in 293T cells.Fluorescent quantitative PCR showed that when PDGFA gene was overexpressed in 293T cells,the expression levels of SPHK2,STAT5A,and STAT5B increased significantly(P<0.01).