Subcellular Localization Of Membrane Fatty Acids Transport Proteins And Oleic Acid Transfer Research In Bovine Mammary Epithelial Cells

The plant-derived long chain fatty acids is the subject to study, and the role of protein mediated transmembrane of long chain fatty acids was researched. First, the coding region of fatty acid transporter-CD36in the cow mammary was cloned byRT-PCR and the sequences were analysed by bioinformatics; Then bovine mammary epithelial cells were separated through tissue culture and collagenase Ⅱ culture; The cell purification was finished by mechanical scraping method and the cytokeratin18and casein was identified by immunofluorescent techniques and RT-PCR, respectively; The GFP fusion plasmid was constructed and the subcellular localization of fatty acid transport proteins in mammary epithelial cell was studied; With the radio labeling technology, the absorption saturation of oleic acid in mammary epithelial cells were studie and the effects of transport inhibitors on the uptake of oleic acid and linoleic acid were researched. The results showed:1. The CD36gene contains a1941bp of open reading frame (ORF) encoding a protein with646amino acid residues; the encoding protein has a relative molecular weight of52.940kD and theoretical isoelectric point of8.36, In comparison with the amino acid sequences of known mammalian CD36, the amino acid sequences deduced from the cDNA sequences of CD36of Chinese Holstein cow showed different levels of homology ranging from82%to91%, respectively; Sequence alignment and phylogenetic tree analysis showed that the relationship of CD36between Bos taurus and Sus scrofa was closest.2. The tissue culture and collagenase Ⅱ culture can separate bovine mammary epithelial cells; The pure epithelial cells were obtained by mechanical scraping method; The cytokeratin18and casein were identified by immunofluorescence and RT-PCR method, which showed that the obtained cells were bovine mammary epithelial cells and have some biological functions.3. Fusion protein of CD36and GFP with pEGFP-C3plasmid was constructed and then transfected into epithelial cells.4. Oleic acid absorption in the mammary epithelial cells showed a saturation of time and concentration dependence. The Michaelis constant (Km) for oleic acid absorption is59.53μM and Vmax is52.63nmol/h/ μg. Membrane transport protein inhibitors-cyclodextrin and Sulfo-N-succinimidyl4-maleimido-oleic acid ester (SSO) significantly inhibited the absorption of oleic and linoleic acids. The uptake saturation of oleic acid and suppression of oleic acid and linoleic acid by inhibitors showed, the membrane proteins-mediated uptake plays an important role during the transport process of long chain fatty acids across epithelial cell membrane.