| 小鼠miRNA-203慢病毒过表达载体的构建及病毒包装与滴度测定 |
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| 引用本文： | 黄学兰,徐广贤,贾伟,王妍柏,董辉,魏军.小鼠miRNA-203慢病毒过表达载体的构建及病毒包装与滴度测定[J].宁夏医科大学学报,2011(7):605-608,F0002. |
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| 作者姓名： | 黄学兰 徐广贤 贾伟 王妍柏 董辉 魏军 |
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| 作者单位： | 宁夏医科大学检验学院;宁夏医科大学附属医院医学实验中心;宁夏大学; |
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| 基金项目： | 国家自然科学基金(30960275); 宁夏自然基金(NZ1079); 宁夏医科大学“博士学位建设学科”开放课题(KF2010-41) |
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| 摘 要： | 目的构建microRNA-203（miR-203）过表达慢病毒载体,并对其进行病毒包装、鉴定与滴度测定。方法利用化学合成miR-203发夹前体结构RNA（small hairpin RNAs,hRNA）,并将其克隆入pSicoR质粒中,经双酶切及测序鉴定;利用脂质体将鉴定的阳性重组pSicoR-miR-203表达载体、pCMV-VSV-G和pCMV-dR8.91三个质粒共转染到HEK-293T细胞,分别在48h和72h收获上清,包装产生慢病毒。将所得病毒悬液梯度稀释后感染293T细胞,检测病毒滴度,并将制备的病毒颗粒尾静脉注射Balb/c小鼠,检测miR-203在小鼠体内的表达与分布情况。结果酶切及测序结果证明成功构建了pSicoR-miR-203重组质粒,并成功的包装成慢病毒,病毒滴度为5×107 TU.mL-1。重组病毒在Balb/c小鼠肝脏、脾脏、肺脏、肾脏均有表达。结论成功构建miR-203慢病毒表达载体,获得高效表达miR-203的慢病毒颗粒,为miR-203靶基因筛选及其功能的研究奠定了基础。
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| 关 键 词： | microRNA-203 慢病毒 绿色荧光蛋白 |
Construction and Identification of Lentiviral Vector Over-expressing Mouse miRNA-203 |
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| HUANG Xue-lan,XU Guang-xian,JIA Wei,WANG Yan-bai,DONG Hui,WEI Jun.Construction and Identification of Lentiviral Vector Over-expressing Mouse miRNA-203[J].Journal of Ningxia Medical College,2011(7):605-608,F0002. |
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| Authors: | HUANG Xue-lan XU Guang-xian JIA Wei WANG Yan-bai DONG Hui WEI Jun |
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| Institution: | HUANG Xue-lan1,XU Guang-xian1,JIA Wei2,WANG Yan-bai1,DONG Hui3,WEI Jun2 (1.Dept.of Examination,Ningxia Med.Univ.,Yinchuan 750004,2.The Clinical Laboratory Center of the General Hospital of Ningxia Med.Univ.,3.Ningxia University,Yinchuan 750004) |
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| Abstract: | Objective To construct a lentiviral vector for the over-expression of microRNA-203(miR-203),and to explore its virus packaging,identification and titer.Methods miR-203 shRNA by chemical synthesis was inserted into pSicoR vector,and then identified by double digestion and nucleotide sequencing.Plasmids pSicoR-miR-203,pCMV-VSV-G and pCMV-dR8.91 were co-transfected into HEK-293T cells for packaging of the lentivirus using liposome,respectively,harvesting the supernatant in 48 hours and 72 hours.HEK-293T cells ... |
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| Keywords: | microRNA-203 lentiviral vector green fluorescent protein |
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