奶牛BTN1A1基因超表达促进乳腺上皮细胞脂滴合成
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摘要
嗜乳脂蛋白(butyrophilin subfamily 1 member A1, BTN1A1)作为一种脂滴蛋白,与乳脂肪球的形成和分泌有关。为研究奶牛(Bos taurus) BTN1A1基因超表达对乳腺上皮细胞脂滴合成的影响,本研究PCR扩增了奶牛BTN1A1基因的CDS,构建Lenti-BTN1A1慢病毒重组载体,用包装好的病毒感染奶牛乳腺上皮细胞,通过嘌呤霉素筛选获得超表达BTN1A1的乳腺上皮细胞株,提取细胞DNA,对超表达细胞株进行基因型鉴定,采用qRT-PCR和Western blot分析细胞株中BTN1A1基因和蛋白的表达;用尼罗红对细胞脂滴进行荧光标记,分析BTN1A1超表达对细胞脂滴的影响,构建BTN1A1-pEGFP-N1载体,观察BTN1A1蛋白与脂滴的共定位。结果显示,克隆的BTN1A1基因CDS区序列大约1 580 bp,成功构建重组的BTN1A1慢病毒载体,用5μg/mL嘌呤霉素筛选得到BTN1A1超表达细胞株,基因型鉴定表明细胞株基因组中成功插入BTN1A1基因;与对照组细胞相比,超表达细胞株的BTN1A1基因mRNA表达增加489倍(P<0.001),蛋白表达也明显增强;检测脂滴的荧光值发现,随着细胞培养时间的延长,BTN1A1超表达显著增加了细胞脂滴含量(P<0.01),共定位发现BTN1A1蛋白主要分布于细胞脂滴上。结果表明,超表达BTN1A1促进乳腺上皮细胞的脂滴合成。本研究为阐明奶牛乳腺上皮细胞的脂滴合成机制提供了基础数据。
As a lipid droplet protein, butyrophilin subfamily 1 member A1(BTN1 A1) is associated with the formation and secretion of milk fat globules. In order to investigate the effect of over-expression bovine(Bos taurus) BTN1 A1 on lipid droplets in mammary epithelial cells, the coding region of BTN1 A1 gene was amplified by PCR, and Lenti-BTN1 A1 lentivirus recombinant vector was constructed. The virus was packaged into recombinant vector and infected bovine mammary epithelial cells. BTN1 A1 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 BTN1 A1 gene and protein was detected by qRT-PCR and Western blot. The effects of BTN1 A1 over-expression on cell lipid droplets were analyzed by fluorescent labeling with Nile red. BTN1 A1-pEGFP-N1 vector was constructed to observe the co-localization of BTN1 A1 protein and lipid droplet. The results showed that the CDS sequence of the cloned BTN1 A1 gene was about 1 580 bp. The lentiviral vector recombinant with BTN1 A1 was constructed. BTN1 A1 cell line was selecteded by 5 μg/m L purinomycin. Genotypic identification showed that exogenous BTN1 A1 gene was successfully inserted into the cell genome. Compared with control group, the expression level of BTN1 A1 gene mRNA was 489 times greater(P<0.001), while protein expression was also increased in over-expression BTN1 A1 cell line. Fluorescence values of lipid droplets showed that over-expression BTN1 A1 significantly increased lipid droplet content with the extension of cell culture time(P<0.01). BTN1 A1 protein was found to co-localized with lipid droplets in cells. In this study, over-expression BTN1 A1 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.
As a lipid droplet protein, butyrophilin subfamily 1 member A1(BTN1 A1) is associated with the formation and secretion of milk fat globules. In order to investigate the effect of over-expression bovine(Bos taurus) BTN1 A1 on lipid droplets in mammary epithelial cells, the coding region of BTN1 A1 gene was amplified by PCR, and Lenti-BTN1 A1 lentivirus recombinant vector was constructed. The virus was packaged into recombinant vector and infected bovine mammary epithelial cells. BTN1 A1 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 BTN1 A1 gene and protein was detected by qRT-PCR and Western blot. The effects of BTN1 A1 over-expression on cell lipid droplets were analyzed by fluorescent labeling with Nile red. BTN1 A1-pEGFP-N1 vector was constructed to observe the co-localization of BTN1 A1 protein and lipid droplet. The results showed that the CDS sequence of the cloned BTN1 A1 gene was about 1 580 bp. The lentiviral vector recombinant with BTN1 A1 was constructed. BTN1 A1 cell line was selecteded by 5 μg/m L purinomycin. Genotypic identification showed that exogenous BTN1 A1 gene was successfully inserted into the cell genome. Compared with control group, the expression level of BTN1 A1 gene mRNA was 489 times greater(P<0.001), while protein expression was also increased in over-expression BTN1 A1 cell line. Fluorescence values of lipid droplets showed that over-expression BTN1 A1 significantly increased lipid droplet content with the extension of cell culture time(P<0.01). BTN1 A1 protein was found to co-localized with lipid droplets in cells. In this study, over-expression BTN1 A1 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.
引文
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张倡珲,郝娟,罗军,等.2017.干扰TIP47基因对奶山羊乳腺上皮细胞脂滴形成及相关基因表达的影响[J].农业生物技术学报,25(2):250-257.(Zhang C H,Hao J,Luo J,et al.2017.Effects of RNAi expression of TIP47 on lipid droplet formation and the expression of related genes in goat(Capra hircus)mammary epithelial cells[J].Journal of Agricultural Biotechnology,25(2):250-257.)
Bionaz M,Loor J J.2008.Gene networks driving bovine milk fat synthesis during the lactation cycle[J].Bmc Genomics,9(1):366.
Brasaemle D L.2007.The perilipin family of structural lipid droplet proteins:Stabilization of lipid droplets and control of lipolysis[J].Journal of Lipid Research,48(12):2547-2559.
Cohen B C,Shamay A,Argov-argaman N.2015.Regulation of lipid droplet size in mammary epithelial cells by remodeling of membrane lipid composition-a potential mechanism[J].PLos One,10(3):e0121645.
Franke W W,Heid H W,Grund C,et al.1981.Antibodies to the major insoluble milk fat globule membrane-associated protein:Specific location in apical regions of lactating epithelial cells[J].The Journal of Cell Biology,89(3):485-494.
Holzmueller W,Kulozik U.2016.Isolation of milk fat globule membrane(MFGM)material by coagulation and diafiltration of buttermilk[J].International Dairy Journal,63:88-91.
Jeong J,Rao A U,Xu J,et al.2009.The PRY/SPRY/B30.2 domain of butyrophilin 1A1(BTN1A1)binds to xanthine oxidoreductase:Implications for the function of BTN1A1 in the mammary gland and other tissues[J].Journal of Biological Chemistry,284(33):22444-22456.
Jeong J,Lisinski I,Kadegowda A K G,et al.2013.A test of current models for the mechanism of milk-lipid droplet secretion[J].Traffic,14(9):974-986.
Lovegrove J A,Givens D I.2016.Dairy food products:Good or bad for cardiometabolic disease?[J].Nutrition Research Reviews,29(2):249-267.
Lucey J A,Otter D,Horne D S,et al.2017.A 100-Year Review:Progress on the chemistry of milk and its components[J].Journal of Dairy Science,100(12):9916-9932.
Mcmanaman J L,Palmer C A,Anderson S,et al.2004.Regulation of milk lipid formation and secretion in the mouse mammary gland[J].Advances in Experimental Medicine&Biology,554:263-279.
Moriya H,Uchida K,Okajima T,et al.2011.Secretion of three enzymes for fatty acid synthesis into mouse milk in association with fat globules,and rapid decrease of the secreted enzymes by treatment with rapamycin[J].Archives of Biochemistry and Biophysics,508(1):87-92.
Nguyen H T H,Ong L,Hoque A,et al.2017.A proteomic characterization shows differences in the milk fat globule membrane of buffalo and bovine milk[J].Food Bioscience,19:7-16.
Ogg S L,Weldon A K,Dobbie L,et al.2004.Expression of butyrophilin(btn1a1)in lactating mammary gland is essential for the regulated secretion of milk-lipid droplets[J].Proceedings of the National Academy of Sciences of the USA,101(27):10084-10089.
Pyc M,Cai,Y Q,Greer,M S,et al.2017.Turning over a new leaf in lipid droplet biology[J].Trends in Plant Science,22(7):596-609.
Reinhardt T A,Lippolis J D.2008.Developmental changes in the milk fat globule membrane proteome during the transition from colostrum to milk[J].Journal of Dairy Science,91(6):2307-2318.
Robenek H,Hofnagel O,Buers I,et al.2006.Butyrophilin controls milk fat globule secretion[J].Proceedings of the National Academy Sciences of the USA,103(27):10385-10390.
Shi H B,Yu K,Luo J,et al.2015.Adipocyte differentiationrelated protein promotes lipid accumulation in goat mammary epithelial cells[J].Journal of Dairy Science,98(10):6954-6964.
Tauchisato K,Ozeki S,Houjou T,et al.2002.The surface of lipid droplets is a phospholipid monolayer with a unique fatty acid composition[J].Journal of Biological Chemistry,277(46):44507-44512.
Wilfling F,Haas J T,Walther T C,et al.2014.Lipid droplet biogenesis[J].Current Opinion in Cell Biology,29:39-45.
Wu C C,Howell K E,Neville M C,et al.2000.Proteomics reveal a link between the endoplasmic reticulum and lipid secretory mechanisms in mammary epithelial cells[J].Electrophoresis,21(16):3470-3482.
张倡珲,郝娟,罗军,等.2017.干扰TIP47基因对奶山羊乳腺上皮细胞脂滴形成及相关基因表达的影响[J].农业生物技术学报,25(2):250-257.(Zhang C H,Hao J,Luo J,et al.2017.Effects of RNAi expression of TIP47 on lipid droplet formation and the expression of related genes in goat(Capra hircus)mammary epithelial cells[J].Journal of Agricultural Biotechnology,25(2):250-257.)
Bionaz M,Loor J J.2008.Gene networks driving bovine milk fat synthesis during the lactation cycle[J].Bmc Genomics,9(1):366.
Brasaemle D L.2007.The perilipin family of structural lipid droplet proteins:Stabilization of lipid droplets and control of lipolysis[J].Journal of Lipid Research,48(12):2547-2559.
Cohen B C,Shamay A,Argov-argaman N.2015.Regulation of lipid droplet size in mammary epithelial cells by remodeling of membrane lipid composition-a potential mechanism[J].PLos One,10(3):e0121645.
Franke W W,Heid H W,Grund C,et al.1981.Antibodies to the major insoluble milk fat globule membrane-associated protein:Specific location in apical regions of lactating epithelial cells[J].The Journal of Cell Biology,89(3):485-494.
Holzmueller W,Kulozik U.2016.Isolation of milk fat globule membrane(MFGM)material by coagulation and diafiltration of buttermilk[J].International Dairy Journal,63:88-91.
Jeong J,Rao A U,Xu J,et al.2009.The PRY/SPRY/B30.2 domain of butyrophilin 1A1(BTN1A1)binds to xanthine oxidoreductase:Implications for the function of BTN1A1 in the mammary gland and other tissues[J].Journal of Biological Chemistry,284(33):22444-22456.
Jeong J,Lisinski I,Kadegowda A K G,et al.2013.A test of current models for the mechanism of milk-lipid droplet secretion[J].Traffic,14(9):974-986.
Lovegrove J A,Givens D I.2016.Dairy food products:Good or bad for cardiometabolic disease?[J].Nutrition Research Reviews,29(2):249-267.
Lucey J A,Otter D,Horne D S,et al.2017.A 100-Year Review:Progress on the chemistry of milk and its components[J].Journal of Dairy Science,100(12):9916-9932.
Mcmanaman J L,Palmer C A,Anderson S,et al.2004.Regulation of milk lipid formation and secretion in the mouse mammary gland[J].Advances in Experimental Medicine&Biology,554:263-279.
Moriya H,Uchida K,Okajima T,et al.2011.Secretion of three enzymes for fatty acid synthesis into mouse milk in association with fat globules,and rapid decrease of the secreted enzymes by treatment with rapamycin[J].Archives of Biochemistry and Biophysics,508(1):87-92.
Nguyen H T H,Ong L,Hoque A,et al.2017.A proteomic characterization shows differences in the milk fat globule membrane of buffalo and bovine milk[J].Food Bioscience,19:7-16.
Ogg S L,Weldon A K,Dobbie L,et al.2004.Expression of butyrophilin(btn1a1)in lactating mammary gland is essential for the regulated secretion of milk-lipid droplets[J].Proceedings of the National Academy of Sciences of the USA,101(27):10084-10089.
Pyc M,Cai,Y Q,Greer,M S,et al.2017.Turning over a new leaf in lipid droplet biology[J].Trends in Plant Science,22(7):596-609.
Reinhardt T A,Lippolis J D.2008.Developmental changes in the milk fat globule membrane proteome during the transition from colostrum to milk[J].Journal of Dairy Science,91(6):2307-2318.
Robenek H,Hofnagel O,Buers I,et al.2006.Butyrophilin controls milk fat globule secretion[J].Proceedings of the National Academy Sciences of the USA,103(27):10385-10390.
Shi H B,Yu K,Luo J,et al.2015.Adipocyte differentiationrelated protein promotes lipid accumulation in goat mammary epithelial cells[J].Journal of Dairy Science,98(10):6954-6964.
Tauchisato K,Ozeki S,Houjou T,et al.2002.The surface of lipid droplets is a phospholipid monolayer with a unique fatty acid composition[J].Journal of Biological Chemistry,277(46):44507-44512.
Wilfling F,Haas J T,Walther T C,et al.2014.Lipid droplet biogenesis[J].Current Opinion in Cell Biology,29:39-45.
Wu C C,Howell K E,Neville M C,et al.2000.Proteomics reveal a link between the endoplasmic reticulum and lipid secretory mechanisms in mammary epithelial cells[J].Electrophoresis,21(16):3470-3482.