State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, PR China.
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, PR China.
Gene. 2014 Apr 25;540(1):110-6. doi: 10.1016/j.gene.2014.02.048. Epub 2014 Feb 26.
α-Tocopherol transfer protein (α-TTP) is a cytosolic protein that plays an important role in regulating concentrations of plasma α-tocopherol (the most bio-active form of vitamin E). Despite the central roles that α-TTP plays in maintaining vitamin E adequacy, we have only recently proved the existence of the α-TTP gene in sheep and, for the first time, cloned its full-length cDNA. However, the study of sheep α-TTP is still in its infancy. In the present study, thirty-five local male lambs of Tan sheep with similar initial body weight were randomly divided into five groups and fed with diets supplemented with 0 (control group), 20, 100, 200, 2000IU·sheep(-1)·d(-1) vitamin E for 120 days. At the end of the experiment, the plasma and liver vitamin E contents were analyzed first and then α-TTP mRNA and protein expression levels were determined by quantitative real-time PCR (qRT-PCR) and Western-blot analysis, respectively. In addition, as no sheep α-TTP antibody was available, a specific monoclonal antibody (McAb) against the ovine α-TTP protein was prepared. The effect of vitamin E supplementation was confirmed by the significant changes in the concentrations of vitamin E in the plasma and liver. As shown by qRT-PCR and Western-blot analysis, dietary vitamin E does not affect sheep α-TTP gene expression, except for high levels of vitamin E supplementation, which significantly increased expression at the protein level. Importantly, the specific sheep anti-α-TTP McAb we generated could provide optimal recognition in ELISA, Western-blot and immunohistochemistry assays, which will be a powerful tool in future studies of the biological functions of sheep α-TTP.
α-生育酚转移蛋白（α-TTP）是一种细胞溶质蛋白，在调节血浆α-生育酚（维生素 E 的最具生物活性形式）浓度方面发挥着重要作用。尽管α-TTP 在维持维生素 E 充足方面起着核心作用，但我们最近才证明绵羊中存在α-TTP 基因，并首次克隆了其全长 cDNA。然而，对绵羊α-TTP 的研究仍处于起步阶段。在本研究中，将 35 只初始体重相似的滩羊本地公羔羊随机分为五组，分别用添加 0（对照组）、20、100、200、2000IU·羊（-1）·d（-1）维生素 E 的日粮进行 120 天的饲养。实验结束时，首先分析血浆和肝脏中维生素 E 的含量，然后通过定量实时 PCR（qRT-PCR）和 Western-blot 分析分别测定α-TTP mRNA 和蛋白表达水平。此外，由于没有绵羊α-TTP 抗体，我们制备了针对绵羊α-TTP 蛋白的特异性单克隆抗体（McAb）。通过血浆和肝脏中维生素 E 浓度的显著变化证实了维生素 E 补充的效果。qRT-PCR 和 Western-blot 分析表明，除了高水平的维生素 E 补充外，日粮维生素 E 不影响绵羊α-TTP 基因表达，高水平的维生素 E 补充会显著增加蛋白水平的表达。重要的是，我们生成的特异性绵羊抗-α-TTP McAb 在 ELISA、Western-blot 和免疫组织化学检测中提供了最佳识别，这将是未来研究绵羊α-TTP 生物学功能的有力工具。
