Zheng H L, Liu X M, Yu Q, Xing R F, Yang Z Y, Luo J
College of Animal Science and Technology, Northwest Agriculture and Forestry University, Key Laboratory of Molecular Biology for Agriculture, Shaanxi Province, Yangling, China.
Genet Mol Res. 2013 Sep 4;12(3):3363-74. doi: 10.4238/2013.September.4.2.
Parathyroid hormone-related protein (PTHrP) is a protein member of the parathyroid hormone family that regulates the dynamic balance between blood and bone calcium during lactation. However, the mechanism of its regulation is not very clear. In order to establish a framework for further functional studies of the PTHrP gene in goat mammary gland epithelial cells during the lactation period, PTHrP cDNA was isolated from Xinong Saanen dairy goats. Its coding sequence is 534 bp in size. We also designed a short hairpin RNA (shRNA) to efficiently inhibit PTHrP expression and constructed recombinant adenoviruses carrying a template encoding this shRNA (AD-PTHrP-322) using the Block-iT shRNA interference system. Finally, the inhibition of PTHrP expression by the recombinant adenoviruses was confirmed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blotting. qRT-PCR results showed that the expression of PTHrP mRNA in mammary epithelial cells was downregulated by 29.2, 68.1, and 82.6% 24, 48, and 72 h after the cells were infected with AD-PTHrP-322, respectively. Western blotting also showed that the expression of PTHrP was reduced in a time-dependent manner. These results suggest that AD-PTHrP-322 significantly inhibits the expression of PTHrP.
甲状旁腺激素相关蛋白（PTHrP）是甲状旁腺激素家族的一个蛋白质成员，在哺乳期调节血液和骨骼钙之间的动态平衡。然而，其调节机制尚不完全清楚。为了建立哺乳期山羊乳腺上皮细胞中PTHrP基因进一步功能研究的框架，从西农萨能奶山羊中分离出PTHrP cDNA。其编码序列大小为534 bp。我们还设计了一种短发夹RNA（shRNA）以有效抑制PTHrP表达，并使用Block-iT shRNA干扰系统构建了携带编码该shRNA模板的重组腺病毒（AD-PTHrP-322）。最后，通过定量逆转录聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法证实了重组腺病毒对PTHrP表达的抑制作用。qRT-PCR结果显示，用AD-PTHrP-322感染细胞后24、48和72小时，乳腺上皮细胞中PTHrP mRNA的表达分别下调了29.2%、68.1%和82.6%。蛋白质免疫印迹法也显示PTHrP的表达呈时间依赖性降低。这些结果表明AD-PTHrP-322显著抑制PTHrP的表达。
