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

作　　者：邢瑞芳[1] 郑惠玲[1] 刘雪梅[1] 闫林慧[1] 安俊辉[1] 杨振宇[1] 祝珍珍[1] 

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100

出　　处：《生物工程学报》2011年第11期1555-1563,共9页Chinese Journal of Biotechnology

基　　金：转基因生物新品种培育重大专项(No.2009ZX08009-162B);中央高校基本科研业务费专项资金(No.Z109021002);陕西省自然基金(No.K332021011)资助~~

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。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%（P0.05）,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.

关 键 词：山羊 甲状旁腺激素相关蛋白 RNA干扰 腺病毒 

分 类 号：S827[农业科学—畜牧学] Q78[农业科学—畜牧兽医]