miR-20b慢病毒表达载体的构建及表达  

Construction and expression of lentiviral vector carrying miR-20b

作　　者：朱莎莎[1] 朱春[2] 余章斌[2] 韩树萍[2] 朱金改[2] 彭宇竹[1] 

机构地区：[1]南京医科大学,江苏省210029 [2]南京医科大学附属南京市妇幼保健院儿科

出　　处：《江苏医药》2014年第2期129-131,F0003,共4页Jiangsu Medical Journal

基　　金：国家自然科学基金(81200126);江苏省医学重点人才基金(RC2011021)

摘　　要：目的构建鼠miR-20b慢病毒表达载体,检测其在P19细胞中的过表达效果。方法设计并合成含有miR-20b成熟序列的编码短发卡状RNA(shRNA)的双链DNA。将shRNA克隆入慢病毒载体,与Pcgvp、Rev和Vsvg包装质粒共转染HEK-293T细胞包装病毒,收取病毒上清液,纯化后感染P19细胞。荧光倒置显微镜下观察感染效率,实时定量PCR检测慢病毒感染后miR-20b的相对表达量。结果经双酶切分析及测序验证,成功构建了miR-20b的过表达载体。慢病毒感染P19细胞的效率可达到70%以上,miR-20b表达水平明显升高。结论成功构建了鼠miR-20b的慢病毒表达载体,包被的病毒可以在P19细胞中实现过表达效果。Objective To construct a recombinant lentiviral vector carrying miR-20b and validate its efficiency in P19 cells. Methods The double-stranded DNA of encoding short hairpin RNA containing mature sequence of miR-20b was designed, synthesized and cloned into a lentiviral vector. The recombinant lentiviral vector and packaging plasmids including Pcgvp, Rev and Vsvg were co-transfected into HEK-293T cells. Then P19 ceils were infected with the supernatant containing lentiviral particles, and its infection efficiency and miR-20b expression were determined by fluorescent microscope and real-time quantitative PCR, respectively. Results The double enzyme digestion and DNA sequencing analysis revealed that the lentiviral vector carrying miR-20b was successfully constructed. The infection efficiency in P19 cells reached over 70 %, and the relative expression level of miR-20b was significantly upregulated. Conclusion The lentiviral vector containing miR-20b with high overexpression in infected P19 cells has been successfully constructed.

关 键 词：慢病毒 P19细胞 

分 类 号：R394[医药卫生—医学遗传学]