拟蜘蛛牵丝蛋白基因细毛羊毛囊特异表达载体的构建及其转基因细胞株的筛选

Construction of Follicle-specific Expression Vector of Spider Dragline Silk Protein and Screening of the Transgenic Cell Lines

试验旨在获得具有毛囊表达特性的转蜘蛛牵丝蛋白基因细胞株。根据GenBank上发表的棒络新妇属蜘蛛的cDNA片段合成拟蜘蛛牵丝蛋白基因单体S,加倍后连入pEGFP-N1框架载体以构建真核表达载体pK-2S,转染新疆美利奴细毛羊皮肤成纤维细胞后筛选单克隆,并通过PCR在DNA、RNA水平检测阳性克隆。基因合成后测序结果显示序列正确,酶切得到正确目的条带,筛选出阳性克隆并且可以在基因组及cDNA上扩增出目的片段。本研究成功将蜘蛛牵丝蛋白基因转入新疆美利奴细毛羊细胞,并在RNA水平表达,为培育毛囊特异表达蜘蛛牵丝蛋白基因并具有更高机械性能羊毛的新型细毛羊品种奠定基础。

This study aimed at getting transgenic cell lines which could express spider dragline silk protein in the hair follicles specifically. By synthesizing spider dragline silk protein gene monomer artificially according to the published eDNA fragments of Nephila elavata spider, got the diploid （2S） by doubling, then constructed hair follicles specific expression vector pK- 2S using pEGFP-N1 as original vector and transfected Xinjiang fine wool merino sheep fetal skin fibroblasts. Make use of PCR to detect positive clones at DNA and RNA level. The sequencing result showed correct sequence after gene synthesis,after enzyme digestion of the vector, we got the correct band while we could amplify target gene from the genom and eDNA of positive clones. This study established cell lines of transgenic spider dragline silk like protein gene successfully while the gene had expressed at RNA level. Our study laid the foundation of making the spider dragline silk gene specifically expressed in the hair follicles of the sheep by transgenic technology.