牛SREBP1基因shRNA序列的筛选及其腺病毒载体的构建与鉴定

【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。

Selection of Effective SREBP1 shRNA in Cattle and the Construction of Recombinant Adenovirus Vector

【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.