牛SREBP1基因shRNA序列的筛选及其腺病毒载体的构建与鉴定  

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

作　　者：付常振[1] 昝林森[1,2] 王虹[1] 成功[1,2] 王洪宝[1,2] 李耀坤[1] 姜碧杰[1] 高建斌[1] 杨宁[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100 [2]国家肉牛改良中心,陕西杨凌712100

出　　处：《中国农业科学》2013年第23期5026-5036,共11页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31272411);"十二五"国家863计划(2011AA100307-02);教育部"长江学者和创新团队发展计划"(IRT0940);"十二五"国家科技支撑计划(2011BAD28B04-03);国家转基因生物新品种培育重大专项(2011ZX08007-002);国家肉牛牦牛产业技术体系(CARS-38)

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(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。[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.

关 键 词：牛 SREBP1 SHRNA 重组腺病毒 

分 类 号：S823[农业科学—畜牧学]