The Site-specific Integration Of Lysostaphin Gene By φC31Integrase In New Born Bovine Ear Fibroblasts

Mastitis is a common problem for lactating cows resulting in decreased milk productionand capabality of reproduction.It is an important problem that protect and cure this disease inthe the livestock industry.breeding the transgenic bovine which is resistent to Mastitis providea new mothed for solving the problem.So the recombination lysostaphin is the target gene inthis project and was inserted into bovine genome by methods of фC31integrase,followingcell transfection and screen.At last,we get the positive cloning,providing donor cell forproduction of transgentic bovine.(1)Construction of vector pEGFP-C1MABTwo same directional loxP elements,β-casein promoter element,recombinant lysostaphinwere inserted into the vector based on the vector pEGFP-C1.Among this elements, Two samedirectional loxP elements were located on the two sides of neorgene.Two672bpchickenβ-lactoglobulin insulators were inserted into the vector.(2) Transfection and screen of bovine ear fibroblastWe got the bovine ear fibroblasts by the tissue explants methods.After Fugene HPtransfection and selection under600μg/mL of G418,we got positive cells with normalform,cell cycle and karyotype.PCR and Southern blotting indentified recombinationlysostaphin has been inserted into genome successfully.(3)Expression of recombinant lysostaphin in Bovine Mammary Epithethial CellWe got the bovine mammary epithethial cell by the tissue explants methods. Afterelectrotransfection and selection under300μg/mL of G418, we got positive cells with normalform.The EGFP was expressed as normal.Western blotting indentified recombinationlysostaphin was expressed successfully.The above results indicate that the vector in the study were inserted into genome ofbovine in a single copy after transfection and selection of Bovine Mammary Epithethial Celland bovine ear fibroblast.This study provide the doner cells for the anti-mastitis transgenicboovine,privide a foundation for the reproduction of anti-mastitis transgenic boovine.