In order to obtain transgenic mouse fibroblast cell line, UHS promoter and polymerized spider dragline silk protein gene(2S) were linked with eukaryotic expression vector pIRES2-EGFP successively, and UHS-2S-pIRES2-EGFP was obtained. Mouse fibroblasts were transfected with the linear plasmid by cationic li-posome method and G418-resistant cells wee cultured. After PCR identified, transgenic cell line with 2S was established. The G418 positive cells were detected, and the results were as follows. 1) UHS-2S-pIRES2-EGFP was constructed successfully. 2) The feasible concentration of G418 was 400 μg/mL in medium. 3) The transgenic cell morphology was similar to normal fibroblast, but the cell growth curve was not of "S" shape, cells proliferated slowly. 4) PCR results showed that the constructed vector was integrated into mouse genome.%为了获得转拟蜘蛛拖丝蛋白基因2S的阳性小鼠成纤维细胞,本研究将前期工作中人工合成的拟蜘蛛拖丝蛋白基因2S和克隆获得的小鼠超高硫角蛋白启动子(UHS),与带有IRES序列和GFP报告基因的表达载体连接,构建真核表达载体UHS-2S-pIRES2-EGFP；将UHS-2S-pIRES2-EGFP线性化后,采用脂质体法转染小鼠皮肤成纤维细胞,通过G418筛选获得neo基因抗性阳性细胞.结果表明:(1)成功构建UHS-2S-pIRES2-EGFP；(2)转基因细胞的最佳G418筛选浓度为400μg/mL；(3)筛选获得呈梭形的转基因细胞,对其进行传代培养时,细胞增殖缓慢,细胞未呈现正常细胞的“S”型的生长曲线；(4) PCR检测结果显示,UHS-2S-pIRES2-EGFP载体已经整合到G418抗性细胞的基因组中.
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