奶山羊LXRα基因shRNA序列的筛选及腺病毒载体的构建与鉴定

Screening of shRNA sequence and construction and identification of recombinant adenovirus vector of LXRα gene of dairy goats

【目的】构建针对奶山羊肝脏X受体α(Liver X receptorα,LXRα)基因的干扰腺病毒载体。【方法】根据奶山羊LXRα基因CDS区域设计小发夹RNA(short hairpin RNA,shRNA),分别构建pENTR/CMV-GFP/U6-shRNA及pDsRed1/C1-LXRα载体,共转染HEK 293细胞,48h后观察其荧光蛋白表达量,筛选出具有明显干扰效应的shRNA。将具有明显干扰效应的shRNA载体与腺病毒骨架载体pAd/PL-DEST在LR ClonaseTMⅡ重组酶的作用下进行重组,经氨苄青霉素及氯霉素抗性筛选后获得重组腺病毒载体。将构建好的重组腺病毒载体经PacⅠ酶切线性化并回收后,转染HEK 293细胞,培养7～10d后收集细胞并反复冻融,收集细胞裂解液,反复扩增3次后,获得高滴度的重组腺病毒,测定其病毒滴度。【结果】①设计出了shRNA-490、shRNA-496、shRNA-509和shRNA-923 4条shRNA序列;②得到shRNA-490和shRNA-923 2条具有明显干扰效应的shRNA;③获得pAd/PL-DEST/CMV-GFP/U6-shRNA-490和pAd/PL-DEST/CMV-GFP/U6-shRNA-923 2个重组腺病毒载体,ScaⅠ酶切及测序结果均正确,2个腺病毒重组载体滴度均为5×108 PFU/mL。【结论】奶山羊LXRα基因重组shRNA腺病毒载体构建成功,为利用RNA干扰技术研究LXRα基因在乳腺上皮细胞中的功能奠定了基础。

【Objective】 The study was to construct the short hairpin RNA（shRNA） recombinant adenovirus vector of dairy goat mammary liver X receptors α（LXRα） gene of Xinong Saanen dairy goat.【Method】 We designed different shRNA targeting the dairy goat LXRα gene CDS region,and constructed pENTR/CMV-GFP/U6-shRNA and pDsRed1/C1-LXRα vectors.The vectors were cotransfected into HEK 293 cells,and the interference efficiency was detected after 48 hours by Fluorescence protein observation.The pENTR/CMV-GFP/U6-shRNA vector and the backbone vector pAd/PL-DEST were recombined under the catalyzation of LR ClonaseTMⅡ,and recombinant vectors were selected by ampicillin chloramphenicol.The recombinant adenovirus vectors were transfected into HEK 293 cell after linearization with PacⅠ enzyme.The cells were frozen and thawed 7-10 days after trasfection,and cell lysates were harvested.Transfection-culture-harvest was repeated three times to obtain high titer of the recombinant adenovirus and detected the titer of the recombinant adenovirus.【Result】 ① Four different shRNA sequences shRNA-490,shRNA-496,shRNA-509 and shRNA-923 were designed.② Two shRNA entry vectors pAd/PL-DEST/CMV-GFP/U6-shRNA-490 and pAd/PL-DEST/CMV-GFP/U6-shRNA-923 showed obvious interference effect.③ScaⅠ enzyme digestion of recombinant adenovirus vector and sequencing analysis were correct with the titer of 5×108 PFU/mL for both vectors.【Conclusion】 Two shRNA recombinant adenovirus vectors were obtained which would play a vital role for the function analysis of LXRα gene in goat primary mammary epithelial cells using RNA interference.