辽宁绒山羊DLX3毛囊表达载体的构建及其转染成纤维细胞的研究

Construction of Hair Follicle Expression Vector of DLX3 and its Transfection into Liaoning Cashmere Goat Fibroblasts Cell

根据GenBank中已发表的DLX3基因(登录号:XM_005694267.1)序列设计引物,以辽宁绒山羊毛囊生长期皮肤组织cDNA为模板,采用RT-PCR方法,扩增出DLX3基因的CDS区,连接平末端载体并验证后与真核表达载体pIRES2-EGFP连接。真核表达载体经XhoⅠ和BamHⅠ双酶切鉴定后,通过脂质体法转染绒山羊耳源成纤维细胞,在荧光显微镜下观察细胞中增强型绿色荧光蛋白(EGFP)的表达,并通过RT-PCR和Western blotting技术检测目的基因转录、蛋白质表达情况。结果表明,成功克隆了绒山羊DLX3基因,并构建了真核表达载体pIRES2-EGFP-DLX3。重组质粒转染绒山羊成纤维细胞24h后在荧光显微镜下观察到绿色荧光,通过RT-PCR扩增909bp的转录产物,并利用Western blotting检测到32.87ku目的蛋白DLX3的表达。本试验结果为研究DLX3基因在绒山羊毛囊生长周期内的功能以及调控毛囊生长发育的机制奠定了基础。

The complete CDS of DLX3 gene was amplified from skin during Liaoning cashmere goat hair follicle growth phase by RT-PCR using one pair of primer which was designed and synthesized according to the DLX3 gene sequence in GenBank Clvo , XM_005694267. 1） , inserted into blunt-vector after DNA sequencing verification, it was then sub-cloned into eukaryotic expression vector pIRES2-EGFP. After the restriction enzyme （Xho I /BamH I ） digestion of the eukaryotic expression vector and sequencing, the plasmid had been transfected into the goat fibroblasts by Lipofectamine 2000. We also observed the fluorescent under the microscope, examined the transcription vector pIRES2-DLX3-EGFP by RT-PCR and Western blotting. The results showed that we had successfully cloned goat DLX3 gene and constructed eukaryotic expression vector pIRES2-DLX3-EGFP,we could observe the green fluorescent after 24h transfection of the plasmid. Then, we amplified the transcription product of 909 bp by RT-PCR. The targeted protein 32. 87 ku was also detected by Western blotting. This result has paved the way for investigating the influence of DLX3 on hair follicle development and cycling as well as the research of the regulation mechanism of hair follicle growth and development.