Abstract: 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.