山羊PTHrP基因干扰重组腺病毒的制备与鉴定

Preparation and identification of recombinant adenoviruses carrying short hairpin RNA targeting parathyroid hormone related protein of goat

摘要:

甲状旁腺激素相关蛋白 (Parathyroid hormone related protein，PTHrP) 具有广泛生物学功能，对调控钙代谢具有重要作用。采用RNA干扰和重组腺病毒技术，对山羊乳腺上皮细胞中PTHrP基因表达进行沉默，为进一步研究该基因在乳腺上皮细胞中的功能奠定基础。采用BLOCK-iT shRNA腺病毒干扰系统，将设计好的寡聚shRNA-322/357经退火复性后插入穿梭质粒pENTR/CMV-GFP/U6中。经Western blotting检测干扰效率后，选择pENTR/CMV-GFP/U6-322与病毒骨架质粒pAd/PL-DEST进行同源重组，成功获得重组干扰腺病毒载体pAD/PL-DEST/CMV-GFP/U6-322，并转染HEK-293细胞，通过包装、扩增后产生干扰重组腺病毒AD-PTHrP-322，TCID50法测定其滴度为2.0×109 PFU/mL。用AD-PTHrP-322感染山羊原代乳腺上皮细胞(MOI=200)，经实时定量PCR及Western blotting检测表明在病毒感染24 h、48 h和72 h后，山羊乳腺上皮细胞中PTHrP mRNA表达量分别下调了29.2％、68.1％和82.6％ (P＜0.05)，其蛋白表达水平也明显下调，干扰效果明显。

Abstract:

Parathyroid hormone related protein (PTHrP) has important biological functions in calcium metabolism. The aim of this study was to silence the expression of PTHrP by RNA interference and recombinant adenovirus, and to provide a material to investigate the relative functions of PTHrP in goat mammary gland epithelial cell. The Block-iT shRNA interference system was used in this experiment. We designed and synthesized two pairs of complementary single-strand DNA oligonucleotides (shRNA-322/357) targeting two different sites of PTHrP mRNA. Then the oligonucleotides were inserted into shuttle vector pENTR/CMV-GFP/U6. After detection of the interference efficiency by Western blotting, we chose pENTR/CMV-GFP/U6-322 and adenovirus backbone vector pAD/PL-DEST to produce recombinant vector pAD/PL- DEST/CMV-GFP/U6-322. The first generation recombinant adenovirus particles (AD-PTHrP-322) were produced and further amplified by transfecting HEK-293 cells. The titer of the recombinant adenovirus reached 2.0×109 PFU/mL determined by TCID50 assays. The result of real-time quantitative PCR indicated that mRNA expression levels of gene were reduced 29.2%, 68.1% and 82.6% (P＜0.05), respectively, when goat mammary gland epithelial cells were infected with AD-PTHrP-322 after 24, 48 and 72 h, in which PTHrP. Western blotting also showed that the expression of PTHrP was reduced by infecting the cells with AD-PTHrP-322. AD-PTHrP-322 has been proved with significant interference effect on expression of PTHrP.