Prokaryotic Expression Of Thermophilic Glycosidase And Establishment Of Bovine Mammary Epithelial Cell Lines

In order to prepare and detect purified transgenic cell line, thermophilic bacteria glycosides prokaryotic expression vector was constructed using PCR and gene recombinant technology. Additionally, a bovine mammary epithelial cell line was established.The lacS was generated from the eukaryotic expression vector p93713 by PCR, In order to obtain lacS protein expressed in E.coli. The authors constructed the recombinant prokaryotic expression plamsid pASK-IBA2-lacS. The recombinant expression plasmid pASK-IBA2-lacS was transformed into E. coli Rosseta and then induced to express the fusion protein by anhydrotetracycline. Two peptides were designed and coupled with carrier protein KLH. Polycolonal antibody serum were obtained from regular immunization of experimental animals(Rabbits), blood draw and centrifugation. The purified antibody was further evaluated with ELISA and Western blot techniques.In second portion of the research project, bovine mammary epithelial cells were isolated , cultured and established; and further identified by morphological study, by cytokeratin K14, K15 immunofluroscence study; and by other biological features and characteristics. Finally, Western blotting assay showed that untransformed mammary epithelial cells do not contain thermophilic bacteria glycosidase.In summary, successful construction of mammary epithelial cells will be invaluable for future research on transgenic animals and target gene expressions at cell level.