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

Chinese. 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。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HEK 293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HEK 293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。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5×109 PFU/mL。获得了有功能的pAd/PL-DEST/CMV-GFP/U6-shRNA病毒重组子。

English. 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. 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-iTTM-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 PacⅠinto 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. 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 5×109 PFU/mL. We got functional adenoviral recombination of pAd/PL-DEST/CMV-GFP/U6-shRNA.