RESUMO
The antibacterial peptide Cecropin B (CB), isolated from the giant silk moth, has been shown to effectively eliminate bacteria. In this study, the effects of transgenic CB on dairy goat mammary epithelial cells (DGMECs) and dairy goat mammary gland were investigated. The DNA of CB from silkworm was amplified by reverse transcription PCR (RT-PCR) and then fused to the eukaryotic expression vector pECFP-C1. The recombinant plasmid pECFP-Cecropin B (pECFP-CB) was used for the transfection of DGMECs, and the expression of transgenic CB and the antibacterial activity of it were confirmed by western blot and agar diffusion reaction respectively. The stable DGMEC line transfected by pECFP-CB was obtained by screening with G418. In vivo experiment, pECFP-CB was injected into dairy goat mammary gland, and also the expression and antibacterial activity of transgenic CB were confirmed. Results of this study: transgenic CB can be expressed in DGMECs and dairy goat mammary gland, and inhibit the mastitis caused by Staphylococcus aureus.
Assuntos
Antibacterianos/metabolismo , Cabras/metabolismo , Proteínas de Insetos/metabolismo , Glândulas Mamárias Animais/metabolismo , Mastite/prevenção & controle , Animais , Antibacterianos/análise , Antibacterianos/farmacologia , Clonagem Molecular , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Cabras/genética , Proteínas de Insetos/análise , Proteínas de Insetos/genética , Proteínas de Insetos/farmacologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/fisiologia , Mastite/microbiologia , Leite/química , Leite/microbiologia , Plasmídeos , Infecções Estafilocócicas/prevenção & controle , Infecções Estafilocócicas/veterinária , Staphylococcus aureus , TransfecçãoRESUMO
To study milk synthesis in dairy goat mammary gland, we had established an in vitro lactating dairy goat mammary epithelial cell (DGMEC) line. Mammary tissues of Guan Zhong dairy goats at 35 d of lactation were dispersed and cultured in a medium containing epithelial growth factor, insulin-like growth factor-1, insulin transferrin serum, and fetal bovine serum. Epithelial cells were enriched by digesting with 0.25% trypsin repeatedly to remove fibroblast cells and were identified as epithelial origin by staining with antibody against cytokeratine 18. The DGMECs displayed monolayer, cobble-stone, epithelial-like morphology, and formed alveoli-like structures and island monolayer aggregates which were the typical characteristics of mammary epithelial cells. A one-half logarithmically growth curve and cytoplasmic lipid droplets in these cells were observed. In this paper, we also studied the lactating function of DGMECs. Results showed that DGMECs could secrete lactose and ß-casein. Lactating function of the cells had no obvious change after 48 h treated by insulin, while prolactin could obviously raise the secretion of milk proteins and lactose.
