Construction Of Lysostaphin Mammary-Specific Expression Vector And Provide The Transgenic Donor Cells For SCNT

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, 2.8 kb 5' regulatory region and 0.6 kb 3' fanking region of bovineβ-casein were used as regulatory region, 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 mmunofluorescence analysis after inducing by prolactin(1mg/mL). 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, and then preparation and identification of the transgenic embryos. The results showed that the lysostaphin gene was expressed in bovine mammary gland epithelial cells and integrated into bovine fetal fibroblasts genome. It is clearly that the obtained lysostaphin transgenic fibroblast cells can be competent for bovine transgenic cloning.1. Construction of the original expression vector of lysostaphin, we successfully expressed the recombinant lysostaphin in the prokaryotic expression system, through the study of its antibacterial activity in vitro, we found that the re-lysostaphin has a good bactericidal effect in vitro, we confirmed that lysostaphin could be used as the functional gene to product the transferred resistance gene animal.2. Bovineβ-casein protein 5 '(BBC5) and 3 ' (BBC3) sequence were amplified from the genome of bovine by PCR, human growth hormone signal peptide sequence and the mature peptide of lysostaphin was amplified from the plasmid of PELY. Firstly, we connected the sequence of BBC5 and BBC3 to the primary mammary expression vector (PEPB) through double digestion and T4 DNA ligase, the recombinant plasmid was named as PBCT .Secondly, we connected the sequence of Hgh-Lys to the PBCT through the same way, and the recombinant plasmid was named as PBS.Finally, we used the same method to connect the PBS to the pEGFP-C1 and got the bovine mammary gland specific expression vector PEPB. 3. We used the FuGENE HD transfection reagent to transfect the purified endotoxin-free plasmid PEPB into bovine mammary epithelial cells, after been induced by prolactin and immunofluorescence staining and Western blot analysis, the results showed that PEPB could mediate the expression of mature peptide of lysostaphin in bovine mammary cells.4. We transfected the purified endotoxin-free plasmid PEPB into bovine fetal fibroblast cells, and observed the green fluorescence expression after 24 hours. while adding G418 to screening the positive cells.Then extracted the cells genome and tested through PCR and Southern blot, the results showed that the target gene had already been ntegrated into bovine fetal fibroblast cell genome and the positive cells can be used as transgenic nuclear donor cells.5. We selected the clone cells to prepared the transfer embryos through SCNT, then extracted the embryos genome with green fluorescent and amplified the target gene from it by PCR , Finally, we correctly obtained the transfer embryos with lysostaphin inside. It is further informted that we successfully obtained the transgenic nuclear donor cells.