Abstract: The purpose of this study was to prepare lysostaphin transgenic donor cells for somatic cell nuclear transfer by constructing mammaryspecific expression vector and transfecting bovine fetal fibroblasts. In this study，pEGFPC1 was used as the vector backbone, 2.6 kb 5′ regulatory region of and 0.6 kb 3′ flanking region of bovine βcasein were used as regulatory regions, the mammary glandspecific expression vector pEPB was constructed, which included neomycin resistant gene and EGFP reporter gene. After identified by PCR and restrictive enzyme digestion, the pEPB was transfected into the bovine mammary epithelial cells through FuGene HD for 35 times, the transgenic cells were detected by immunofluorescence analysis after induced by prolactin（1 mg·mL-1）. And then, the plasmid pEPB was transfected into the bovine fetal fibroblast cells by electroporation, positive cells were screened by G418 selection and determined by PCR and Southern blot. The results showed that the lysostaphin gene was expressed in bovine mammary gland epithelial cells and integrated into bovine fetal fibroblasts genome. The results indicate that the lysostaphin transgenic fibroblast cells obtained may be competent for bovine transgenic cloning.