Construction of Fibroblast Growth Factor 5 Gene (FGF5) Hair Follicle Specific Expression Vector and Transfected into Cashmere Goat (Capra hircus) Fetal Fibroblast Cells
Abstract The quality and yield of cashmere goat wool are closely related to the growth and development of skin hair follicles. This study aimed to construct an eukaryotic expression vector and prove whether it could specifically express fibroblast growth factor 5 gene (FGF5) in the hair follicles of cashmere goat (Capra hircus). The keratin associated protein 6-1 (KAP6-1) promoter and FGF5 gene coding sequence were obtained by PCR. The vector pEGFP-N1-KF, a hair follicles specific expression vector of FGF5, was constructed by inserting the KAP6-1 promoter into the CMV promoter-less pEGFP-N1, and then connecting FGF5 to the downstream of KAP6-1 promoter, using Porcine teschovirus 2A peptide (P2A) to link FGF5 and enhanced green fluorescent protein (EGFP) gene. The constructed vector was transfected into cashmere goat fetal fibroblast cells and two methods were used to verify its functionality including qRT-PCR and Western blot. The enzyme digestion results showed the vector was correctly constructed. The qRT-PCR results indicated that the FGF5 gene could express in fetal fibroblast cells. The Western blotting results confirmed that the fusion protein could be availably spelited into FGF5 and GFP protein by the functionality of P2A. The result showed that the vector pEGFP-N1-KF which could specifically express FGF5 gene was successfully constructed and could normally expressed in cashmere goat fetal fibroblast cells.
