CAJ | 학술논문

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cDNA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pEGFP-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pEGFP-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qRT-PCR和Western blot检测。酶切鉴定结果表明，载体pEGFP-N1-KF构建成功；qRT-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。
융산양적융모품질、산량여피부모낭적생장발육밀절상관。본연구지재구건융산양(Capra hircus)성섬유세포생장인자5기인(fibroblast growth factor 5, FGF5)적모낭특이성표체재체，병증명기표체적유효성。분별이융산양기인조화cDNA위모판，이용PCR방법극륭각단백관련단백6-1(keratin associated protein 6-1, KAP6-1)기인적계동자화FGF5기인적편마구서렬(coding sequence, CDS)，병장저량개원건련접도거제CMV계동자적진핵표체재체pEGFP-N1상，FGF5여증강록색형광단백(enhanced green fluorescent protein, EGFP)기인표체광지간이저첩신병독(Porcine teschovirus, PTV)2A(P2A)상련，구건모낭특이성표체재체pEGFP-N1-KF。표체재체전염융산양태인성섬유세포후，대세포표체산물진행qRT-PCR화Western blot검측。매절감정결과표명，재체pEGFP-N1-KF구건성공；qRT-PCR결과표명， FGF5정상표체；Western blot결과현시，P2A능구유효전절FGF5화EGFP융합단백。표명성공득도료융산양FGF5기인적모낭특이성표체재체병재융산양태인성섬유세포중능정상표체。본실험위진일보연구융산양FGF5기인재모낭발육화주기조공중적작용제공료기술지지。
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.