中文摘要：

【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（codingsequence，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9×10^8GFU·mL^-1。Ad-1053和hd-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。

英文摘要：

[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 fiLrther inserted into pENTR-U6 to construct pENTR-U6-shRNA expression vector. The cotransfection of expression vector psiCHECK- II carrying 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× 10^8 GFU/mL and 9x 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.