Regulation Effect Analysis Of BTLF Promoter Elements

Lactoferrin (LTF) belongs to transferrin gene family.It is an iron-bindingglycoprotein. The middle of the polypeptide chain is an extended peptide chain, bothends are folded into a sphere, which combined with Fe3+and HCO-3.Lactoferrin wasdetected in milk of bovine and other species. It also exists in many other exocrinesecretions such as saliva, spermatic fluid and tears.In this study, upstream region5’ flanking-6to-1799seqμence of bovinelactoferrin (bLTF) gene was analysed to determine the positive and negativeregulatory elements. Deletions and mutation on negative regulatory element wascarried oμt to obtain a more active promoter. First, transcription factor binding sites ofbLTF upstream5’ flanking-6to-1799were predicted. The first experiment wascarried out based on the predicted result. Different length of promoters were obtainedby piecewise missed gene sequences to replace CMV promoter of pEGFP-N1toconstructe recombinant vectors. Lipofectamine2000transfection technique wasadopted to stabilize transfect bovine mammary epithelial cells and obtain monoclonalcell lines. Finally, fluorescence microscopy, multifμnctional microplate readerquantitative detection and Real-time PCR to detect EGFP expression efficiency in thecells.By statistical data analysis, promoter activity intensity was determined.Thesecond experiment studied negative regulatory element to further validate the resultsof previous experiment by sequence mutation and deletion,and obtained an activepromoter.In the third experiment,-979to-1135(length156bp) was deleted toanalysed its promoter activity.In this study,we analysed the regulation of bLTF gene promoter. Providing amolecular basis for exploring methods of improving bLTF production.