Abstract

Spider silk, which has remarkable characteristics, has wide application prospects in many fields. Many researchers have explored potential methods for directly producing spider silk proteins and spidroins with mechanical properties or obtaining recombinant spider silk fibers by genetic engineering methods. However, there are still some shortcomings with these methods, such as inability to simulate the fibrosis process of spider silk. In this study, a high glycine/tyrosine protein gene (HGT) promoter originate from sheep was first cloned by PCR. The HGT promoter was ligated into pcDNA3.1 and pcDNA3.1-HGT was obtained. After linking with the synthesized and polymerized gene 4S, a eukaryotic expression vector pcDNA3.1-HGT-4S was constructed using a series of molecular methods. Sheep fibroblasts transfected with the linearized plasmid using a liposome-mediated method were screened with G418 and a transgenic cell line was established. Cells from the transgenic line were used as nuclear donors to construct embryos with somatic cell nuclear transfer (SCNT). Reconstructed embryos derived from transgenic cells were able to develop in vitro successfully. PCR was carried out and results demonstrated that the synthetic spidroin gene 4S had integrated into the embryo genome. In summary, we explored a method and successfully obtained artificial synthetic spidroin gene transgenic sheep cloned embryos with a hair follicle specific promoter by SCNT. Further research is necessary on transgenic sheep with synthetic spidroin genes expressed in hair follicles.

摘要

蜘蛛丝具有显着的特性，在许多领域具有广泛的应用前景。许多研究人员探索了直接生产具有机械性能的蜘蛛丝蛋白和蜘蛛蛋白或通过基因工程方法获得重组蜘蛛丝纤维的潜在方法。但是，这些方法仍然存在一些不足，例如无法模拟蜘蛛丝的纤维化过程。在这项研究中，首先通过 PCR 克隆了一个源自绵羊的高甘氨酸/酪氨酸蛋白基因 (HGT) 启动子。将HGT启动子连接到pcDNA3.1中，得到pcDNA3.1-HGT。与合成聚合的基因4S连接后，采用一系列分子方法构建真核表达载体pcDNA3.1-HGT-4S。使用脂质体介导的方法用线性化质粒转染的绵羊成纤维细胞用 G418 进行筛选，并建立了转基因细胞系。来自转基因系的细胞被用作核供体以构建体细胞核移植 (SCNT) 胚胎。来自转基因细胞的重建胚胎能够发育体外成功。进行PCR，结果表明合成蛛丝蛋白基因4S已整合到胚胎基因组中。综上所述，我们探索了一种方法，通过SCNT成功获得了具有毛囊特异性启动子的人工合成蜘蛛蛋白基因转基因绵羊克隆胚胎。需要进一步研究在毛囊中表达合成蜘蛛蛋白基因的转基因绵羊。