| 摘要: |
| 【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，48 h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7～10 d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。 |
| 关键词: 肝脏X受体α RNA干扰 腺病毒载体 奶山羊 |
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| 基金项目:农业部转基因生物新品种培育重大专项（2009ZX08009-162B） |
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| Screening of shRNA sequence and construction and identification of recombinant adenovirus vector of LXRα gene of dairy goats |
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| Abstract: |
| 【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. |
| Key words: liver X receptor α RNA interference adenovirus vector dairy goat |
