| 慢病毒介导的水牛CD14基因shRNA序列的筛选 |
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| 引用本文： | 李美青,康超,黄时海,覃兆鲜,路新梅,乔树叶,李湘萍,石德顺.慢病毒介导的水牛CD14基因shRNA序列的筛选[J].畜牧与兽医,2012,44(12):4-9. |
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| 作者姓名： | 李美青 康超 黄时海 覃兆鲜 路新梅 乔树叶 李湘萍 石德顺 |
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| 作者单位： | 亚热带农业生物资源与保护利用重点国家实验室,广西南宁,530004 |
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| 基金项目： | 国家转基因生物新品种培育重大专项重点课题 |
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| 摘 要： | 研究慢病毒载体介导的RNA干扰对水牛CD14基因表达的影响,设计并筛选具有抑制作用的靶序列。将真核表达载体pDsRed-N1-buffaloCD14转染293细胞株,建立稳定表达水牛CD14基因细胞系。同时设计5条水牛CD14 shRNA(319/421/755/970/1 041)以及1条阴性对照序列(NC-1864),构建慢病毒重组质粒pSicoR-GFP-shRNA。采用磷酸钙沉淀法将三质粒共转染293T细胞包装慢病毒,并进行滴度测定。最后,通过对慢病毒感染的pDsRed1-N1-buffalo CD14-293细胞进行QRT-PCR检测,筛选具有抑制效果的shRNA。结果显示:在各感染细胞组中,外源水牛CD14基因mRNA的表达均受到不同程度的抑制,其中,CD14 shRNA-970靶点的干扰效率最高,达到79.3%(P<0.01)。成功筛选出具有较高抑制效果的水牛CD14 shRNA靶序列,为研究CD14基因在布氏杆菌所致炎症反应中的作用,建立家畜布氏杆菌病防治新方法奠定了基础。
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| 关 键 词： | 布氏杆菌病 水牛CD14 RNA干扰 慢病毒载体 |
Screening of lentiviral-mediated shRNA sequences targeting buffalo CD14 gene |
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| LI Mei-qing
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KANG Chao
,
HUANG Shi-hai
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QIN Zhao-xian
,
LU Xin-mei
,
QIAO Shu-ye
,
LI Xiang-ping
,
SHI De-shun.Screening of lentiviral-mediated shRNA sequences targeting buffalo CD14 gene[J].Animal Husbandry & Veterinary Medicine,2012,44(12):4-9. |
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| Authors: | LI Mei-qing KANG Chao HUANG Shi-hai QIN Zhao-xian LU Xin-mei QIAO Shu-ye LI Xiang-ping SHI De-shun |
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| Institution: | *(State Key Laboratory for Conservation and Utilization of Agricultural Bioresources in the Subtropics, Guangxi University,Nanning 530004,China) |
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| Abstract: | The study is aimed to investigate the effects of lentiviral vector-mediated RNAi on buffalo CD14 gene expression,and to screen the target sequence with inhibition effect.Eukaryotic expression vector pDsRed1-N1-buffalo CD14 was transfected into 293 cells to get the cell lines expressing buffalo CD14 gene.Meanwhile,five CD14 shRNA(319/421/755/970/1 041) sequences and a negative control sequence(NC-1864) were designed,synthesized and constructed into lentiviral recombinant plasmids pSicoR-GFP-shRNA.Lentivirus was obtained by packaging three plasmids into 293T cells and using calcium phosphate precipitation method,and then the titer of virus was tested.Finally,the expression of CD14 mRNA in pDsRed1-N1-buffalo CD14-293 cell lines infected by lentivirus was examined by QRT-PCR.The results showed that the expression of exogenous buffalo CD14 mRNA was reduced at different levels in the infected cell groups.Among the five CD14 shRNA sequences,shRNA-970 had the highest interfering efficiency of 79.3%(P<0.01).This study laid a foundation for further investigation on the role of CD14 in the inflammatory reaction induced by Brucella and the establishment of a new way to cure Brucellosis in livestock. |
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| Keywords: | Brucellosis buffalo CD14 RNA interference Lentviral vector |
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