中文摘要：

为了筛选转pK2.10-拟蜘蛛拖丝蛋白基因绵羊成纤维细胞株，本研究参照GenBank上蜘蛛拖丝蛋白基因序列（AY555585和AH015065）设计并人工合成拟蜘蛛基因四聚体（4S），与pcDNA3.1载体（K2.10启动子）相连构建真核表达载体（pcDNA3.1-K2.10-4S），采用脂质体介导法转染绵羊成纤维细胞，通过药物筛选、阳性细胞鉴定及体外培养等方法，获得了转蜘蛛拖丝蛋白基因的绵羊成纤维细胞株。结果，用pcDNA3.1-K2.10-4S表达载体转染冷冻复苏的绵羊成纤维细胞后，利用G418筛选法获得neo基因的抗性细胞，即阳性细胞；采用PCR方法对阳性细胞鉴定显示，所构建的载体整合到了细胞基因组中；对阳性细胞的染色体核型分析显示，阳性细胞核型稳定，与正常细胞的核型无差异；阳性细胞经冷冻复苏后仍能保持原有的特性。该细胞株的建立为进一步开展转拟蜘蛛拖丝蛋白基因的克隆羊研究奠定了基础。

英文摘要：

According to spider dragline silk protein gene sequence in GenBank（AY555585 and AH015065）,tetra-polymerized silk protein gene（4S） was artificially synthesized. Eukaryotic expression vector was constructed by linking the 4S to pcDNA3.1-K2.10（hair follicle-specific promoter）. Sheep fibroblasts were transfected with the plasmid by cationic liposome method and filtrated by using G418. After identified, transgenic cell line with spider dragline silk protein gene was established. Results showed that sheep skin fibroblasts were isolated from fresh and were transfected with linearization vector. Using G418 to select cells, G418 resistance cells were gained. G418 resistance cells were identified by PCR method, the result showed that the vector constructed was integrated into sheep genome. At the same time, chromosome of G418 resistance cell was analysed, the result showed that chromosome of these cells were stable and there were no different between these cells and normal cells. The transgenic sheep fibroblast cell line with artificial synthesized spider dragline silk protein gene would be used in the future transgenic sheep study.