绒山羊成纤维细胞生长因子5基因(FGF5)毛囊特异性表达载体的构建及转染胎儿成纤维细胞  

Construction of Fibroblast Growth Factor 5 Gene (FGF5) Hair Follicle Specific Expression Vector and Transfected into Cashmere Goat (Capra hircus) Fetal Fibroblast Cells

作　　者：康健[1] 胡广东[1] 权富生[1] 张涌[1] 

机构地区：[1]西北农林科技大学动物医学院/农业部动物生物技术重点实验室,杨凌712100

出　　处：《农业生物技术学报》2014年第12期1536-1543,共8页Journal of Agricultural Biotechnology

基　　金：国家高科技研究发展计划(863计划)(No.2011AA100303)

摘　　要：绒山羊的绒毛品质、产量与皮肤毛囊的生长发育密切相关。本研究旨在构建绒山羊(Capra hircus)成纤维细胞生长因子5基因(fibroblast growth factor 5,FGF5)的毛囊特异性表达载体,并证明其表达的有效性。分别以绒山羊基因组和c DNA为模板,利用PCR方法克隆角蛋白关联蛋白6-1(keratin associated protein 6-1,KAP6-1)基因的启动子和FGF5基因的编码区序列(coding sequence,CDS),并将这两个元件连接到去除CMV启动子的真核表达载体p EGFP-N1上,FGF5与增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因表达框之间以猪捷申病毒(Porcine teschovirus,PTV)2A(P2A)相连,构建毛囊特异性表达载体p EGFP-N1-KF。表达载体转染绒山羊胎儿成纤维细胞后,对细胞表达产物进行q RT-PCR和Western blot检测。酶切鉴定结果表明,载体p EGFP-N1-KF构建成功;q RT-PCR结果表明,FGF5正常表达;Western blot结果显示,P2A能够有效剪切FGF5和EGFP融合蛋白。表明成功得到了绒山羊FGF5基因的毛囊特异性表达载体并在绒山羊胎儿成纤维细胞中能正常表达。本实验为进一步研究绒山羊FGF5基因在毛囊发育和周期调控中的作用提供了技术支持。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.

关 键 词：FGF5 载体构建 胎儿成纤维细胞 绒山羊 

分 类 号：S827[农业科学—畜牧学]