牛SREBP1基因shRNA序列的筛选及其腺病毒载体的构建与鉴定

摘要

[Objective] The aim of this study was to construct recombinant adenovirus carrying effective small hairpin RNA (shRNA) which can exclusively interfere NotI bovine sterol-regulatory-element-binding protein 1 (SREBP1) gene expression, thus providing a basis for studying the function and mechanism of SREBP1 gene at cellular level.[Method]According to the coding sequence (CDS) region of SREBP1 gene, six pairs of inhibition shRNA and one pairs of negative control shRNA were designed and further inserted into pENTR-U6 to construct pENTR-U6-shRNA expression vector. The cotransfection of expression vector psiCHECK-Ⅱcarrying SREBP1 and the obtained pENTR-U6-shRNA was carried out in 293 A cell lines to select efficient shRNA. The efficient shRNA and shRNA-NC were connected to pAD/BL-DEST to construct the recombinant plasmid, respectively. The obtained recombinant adenovirus vectors were transfected into 293A cells to package. Then, the adenovirus were amplified and harvested. Real-time PCR (qRT-PCR) was used to detect and confirm the interference effect of the harvested adenovirus on the target gene SREBP1 in bovine pre-adipocytes. The viral titer was determined by GFP labeling method. [Result]Results showed that shRNA-1053 significantly decreased the expression of SREBP1 by 87.4%. The linearized recombinant adenovirus vector carrying shRNA-1053 and shRNA-NC transfected 293A cells, and further packaged and amplified high-titer recombinant adenovirus Ad-1053 and Ad-NC (7×108 GFU/mL and 9×108 GFU/mL). qRT-PCR results elucidated Ad-1053 significantly down-regulated mRNA expression level of SREBP1 gene in bovine pre-adipocytes (Interfering efficiency >85%), while Ad-NC did not.[Conclusion]In this study, the recombinant adenovirus, carrying efficient shRNA designed for RNA interference study of bovine SREBP1 gene, was constructed successfully.%[目的]筛选可靶向干扰牛的SREBP1基因的有效shRNA，构建相应腺病毒载体并包装重组腺病毒，为在细胞水平上研究SREBP1基因的功能和作用机制提供基础。[方法]以秦川牛SREBP1基因为研究对象，首先靶向其编码区（coding sequence，CDS）序列设计并合成6条干扰和1条阴性对照shRNA，并构建表达载体pENTR-U6-shRNA。然后与载体psiCHECK-Ⅱ-SREBP1共转染293A细胞，筛选有效的pENTR-U6-shRNA。其次将筛选的pENTR-U6-shRNA和阴性对照pENTR-U6-NC分别与腺病毒骨架载体pAd/PL-DEST体外重组，得到腺病毒重组载体，并在293A细胞包装扩繁得到高滴度腺病毒，GFP标记法测定病毒滴度。最后将病毒侵染牛前体脂肪细胞，实时荧光定量法（qRT-PCR）检测干扰效率。[结果]筛选出靶向牛SREBP1基因干扰效率为87.4%的shRNA-1053。构建了pAd-1053和阴性对照pAd-NC重组腺病毒载体并包装得到Ad-1053和Ad-NC高滴度病毒，测定得到的病毒滴度分别为7×108 GFU·mL-1和9×108 GFU·mL-1。Ad-1053和Ad-NC分别侵染牛前体脂肪细胞，qRT-PCR检测结果显示Ad-1053可显著降低SREBP1基因的mRNA 水平（干扰率＞85%），而Ad-NC对SREBP1基因表达无明显影响。[结论]成功筛选得到靶向干扰牛SREBP1基因的有效shRNA，并包装扩繁获得相应的高滴度重组病毒。