西农萨能羊BTN基因RNA干扰序列的筛选及重组腺病毒载体的构建与鉴定  

Screening of RNA interference sequence and construction and identification of BTN recombinant adenovirus of Xinong Saanen Goat

作　　者：赵旺生[1] 罗军[1] 王伟[1] 滕炎玲[1] 王桢[1] 林先滋[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西省农业分子生物学重点实验室,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2010年第12期38-44,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：转基因生物新品种培育项目(2009ZX08009-162B);公益性行业(农业)专项科研经费项目(3-45);陕西省重大科技创新项目(2009ZKC07-01-1)

摘　　要：【目的】构建西农萨能羊BTN基因RNA干扰的重组腺病毒载体,为研究BTN基因的功能和作用机制奠定基础。【方法】设计并合成3对针对BTN不同位点的shRNA编码序列,克隆到pENTR/CMV-GFP/U6-shRNA载体中,并与已构建好的表达BTN的pDsRed1-C1-BTN载体共转染HEK293细胞,筛选有效的干扰序列。通过与腺病毒骨架质粒重组,制备表达干扰BTN基因的shRNA的腺病毒,经PacⅠ线性化后,在HEK293细胞中包装并扩增病毒,用TCID50法进行病毒滴度测定,获得高滴度的病毒上清液。【结果】成功构建了西农萨能羊BTN基因的RNAi腺病毒表达载体,腺病毒经包装和扩增后,病毒滴度可达到5×109PFU/mL。【结论】获得了有功能的pAd/PL-DEST/CMV-GFP/U6-shRNA病毒重组子。[Objective] The aim of this project was to construct RNAi recombinant adenoviral vector specific to BTN gene and to establish foundation on function and mechanism of BTN gene.[Method] The first step was to design and synthesize three pairs of complementary single-strand DNA oligos targeting three various sites of BTN mRNA,and then oligos were cloned into pENTR/CMV-GFP/U6-shRNA,the entry vector,to generate the entry clone to co-transfect HEK 293 cells with pDsRed1-C1-BTN expressing BTN gene to select effective RNAi sequence.Recombination reaction in vitro with the pENTR and pAd/BLOCK-iT^TM-DEST,the adenovirus backbone vector,were used to creat the adenovirus plasmid which expressed the interference of BTN gene.Then,we transfected the adenovirus plasmid digested with Pac Ⅰ into HEK 293 cells to produce adenovirus,and infected the 293 cells with the crude adenovirus to amply the adenoviral stock.TCID50 assay was used to titer the adenoviral stock and got a high titer.[Result] The RNAi adenovirus exppression vector targeting to BTN gene of Xinong Saanen Goat was constructed successfully,after being packaged and amplified,the titer of the adenovirus can reach 5×10^9 PFU/mL.[Conclusion] We got functional adenoviral recombination of pAd/PL-DEST/CMV-GFP/U6-shRNA.

关 键 词：嗜乳脂蛋白基因 RNA干扰 腺病毒载体 

分 类 号：S826.94[农业科学—畜牧学] Q782[农业科学—畜牧兽医]