Over-expression of Bovine (Bos taurus) BTN1A1 Gene Promotes Lipid Droplet Synthesis in Mammary Epithelial Cells
ZHANG Meng-Lu*, FAN Xiao-Na*, XING Zhi-Yang, WANG Yue-Ying, WANG Jiang, CHU Bei-Bei, HAN Li-Qiang**, YANG Guo-Yu**
College of Animal Science and Veterinary Medicine/Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture, Henan Agricultural University, Zhengzhou 450002, China
Abstract：As a lipid droplet protein, butyrophilin subfamily 1 member A1 (BTN1A1) is associated with the formation and secretion of milk fat globules.In order to investigate the effect of over-expression bovine (Bos taurus) BTN1A1 on lipid droplets in mammary epithelial cells, the coding region of BTN1A1 gene was amplified by PCR, and Lenti-BTN1A1 lentivirus recombinant vector was constructed.The virus was packaged into recombinant vector and infected bovine mammary epithelial cells.BTN1A1 over-expression cell lines were selected by applying puromycin in cell culture medium.DNA was extracted from the over-expressed cell lines and genotypes were identified.Expression of BTN1A1 gene and protein was detected by qRT-PCR and Western blot.The effects of BTN1A1 over-expression on cell lipid droplets were analyzed by fluorescent labeling with Nile red.BTN1A1-pEGFP-N1 vector was constructed to observe the co-localization of BTN1A1 protein and lipid droplet.The results showed that the CDS sequence of the cloned BTN1A1 gene was about 1 580 bp.The lentiviral vector recombinant with BTN1A1 was constructed.BTN1A1 cell line was selecteded by 5 μg/mL purinomycin.Genotypic identification showed that exogenous BTN1A1 gene was successfully inserted into the cell genome.Compared with control group, the expression level of BTN1A1 gene mRNA was 489 times greater (P<0.001) ,while protein expression was also increased in over-expression BTN1A1 cell line.Fluorescence values of lipid droplets showed that over-expression BTN1A1 significantly increased lipid droplet content with the extension of cell culture time (P<0.01).BTN1A1 protein was found to co-localized with lipid droplets in cells.In this study, over-expression BTN1A1 promoted lipid droplet synthesis in mammary epithelial cells.This study provides basic data to elucidate the mechanism of lipid droplet synthesis in mammary epithalial cells of dairy cows.
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