真核表达载体pcDNA3.1(+)-GFP-MC1R的构建及在羊驼毛囊干细胞中的表达

Construction of eukaryotic expression vector pcDNA3.1(+)-GFPMC1R and its expression in alpaca hair follicle stem cells

旨在构建含有MC1R基因的pcDNA3.1(+)真核重组表达载体,为进一步研究MC1R的生物学功能奠定基础。应用RT-PCR技术从羊驼皮肤cDNA文库中扩增MC1R基因全长cDNA,然后通过双酶切将MC1R基因全长cDNA克隆到真核表达载体pcDNA3.1(+),然后采用脂质体法转染体外培养的羊驼毛囊干细胞后,通过G418筛选培养,建立稳定的转染体系,直接荧光观察pcDNA3.1/MC1R融合蛋白在细胞中的分布定位,并通过Western Blot方法检测MC1R在羊驼毛囊干细胞中的表达。结果表明,成功构建了pcDNA3.1/MC1R重组表达载体,并且发现试验组的毛囊干细胞中表达MC1R蛋白显著高于空白组(P<0.05)。成功构建了含有绿色荧光蛋白基因的真核表达载体pcDNA3.1/MC1R,且MC1R基因在羊驼毛囊干细胞中获得表达。

It is the base of further study on MC1R biological functions to construct the pcDNA3 .1 eukaryotic expression vector of MC1R gene in alpaca .MC1R gene was amplified by RTPCR with the cDNA from alpaca skin library .PCR products and pcDNA3 .1 plasmid were digested and recycled by BamHI and EcoRI endonuclease .The positive clones were selected , from which plasmid DNA was abstracted and identified by restriction endonuclease ,sequence identification and sequencing . The pcDNA3 .1/MC1R recombinant expression plasmid was transfected into alpaca hair follicle stem cells by Lipofectamine TM 2000 .After screening culture by G418 ,a stablytransfected cell system was established .Fluorescent microscopy was employed to reveal the localization of MC1R in alpaca hair follicle stem cells ,and the transcription and expression of the MC1R were identified by Western Blot .The results show that eukaryotic expression vector pcDNA3 .1/MC1R was successfully constructed .When pcDNA3 . 1/MC1R recombinant expression plasmids were transfected into alpaca hair follicle stem cells , the expression of MC1R was upregulated （P〈0 .05） .This study successfully constructed eukaryotic expression vector containing green fluorescent protein gene pcDNA 3 .1/MC1R .It could upregulate the expression of MC1R in alpaca hair follicle stem cells .