上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

Porf-2基因过表达慢病毒载体的构建以及神经细胞的转染

黄国辉1，杨西涛1，陈奎1，邢进1，朱亮1，李泓江1，冯东福1,2   

  1. 上海交通大学 医学院1.附属第三人民医院神经外科， 上海 201900； 2.创伤医学研究所， 上海 201900
  • 出版日期:2015-12-28 发布日期:2016-01-21
  • 通讯作者: 冯东福, 电子信箱: dffeng@21cn.com。
  • 作者简介:黄国辉（1990—）， 男， 博士生; 电子信箱: huangheidou@163.com。
  • 基金资助:

    国家自然科学基金项目（81171796）。

Construction of lentiviral vector with over-expression of Porf-2 gene and transfection of neural cells

HUANG Guo-hui1, YANG Xi-tao1, CHEN Kui1 , XING Jin1, ZHU Liang1, LI Hong-jiang1, FENG Dong-fu1,2   

  1. 1.Department of Neurosurgery, Shanghai Third Peoples Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai  201900,China; 2.Institute of Traumatic Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 201900, China
  • Online:2015-12-28 Published:2016-01-21
  • Supported by:

    National Natural Science Foundation of China, 81171796

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摘要：

目的  构建视前区调控因子2（Porf-2）与绿色荧光蛋白（GFP）共表达的慢病毒载体，包装慢病毒并体外感染神经细胞。方法  根据GenBank中基因信息，设计Porf-2的引物，采用PCR扩增Porf-2基因片段；运用基因重组技术,双酶切Porf-2，并将其克隆至pLVX-IRES-ZsGreen1载体，经酶切、测序加以鉴定重组质粒。将成功构建的质粒与包装系统VSVG 、Δ89质粒共转染293T细胞，包装慢病毒并测定滴度。结果  经酶切和测序鉴定表明成功构建了pLVX-Porf-2-IRES-ZsGreen1的基因重组慢病毒载体；将成功包装的病毒感染NG108细胞、海马原代神经元以及神经干细胞后，可观察到大量绿色荧光表达；Western blotting证实Porf-2成功过表达。结论  成功构建Porf-2与GFP基因共表达的慢病毒载体、包装慢病毒，并感染NG108、海马原代神经元以及神经干细胞。

关键词: 视前区调控因子2, 绿色荧光蛋白, 慢病毒, 神经细胞

Abstract:

Objective  To construct a co-expressing lentiviral vector of preoptic regulatory factor-2 (Porf-2) and green fluorescent protein (GFP), package the lentiviruses, and transfect neural cells in vitro. Methods  Primers of Porf-2 were designed according to the gene information of GenBank. Gene fragments of Porf-2 were amplified by polymerase chain reaction (PCR). By using the gene recombinant technology, Porf-2 was double digested and cloned to pLVX-IRES-ZsGreen1 vector. Recombined plasmids were identified by enzyme digestion and DNA sequencing. The 293T cells were transfected with constructed plasmids, packaging system VSVG, and Δ89 plasmids. Lentiviruses were packaged and the titer was determined. Results  Enzyme digestion and DNA sequencing confirmed that the gene recombinant lentiviral vector of pLvx-Porf-2-IRES-ZsGreen1 had been successfully constructed. After NG108, primary hippocampal neurons, and neural stem cells were transfected by the packaged lentiviruses, a large number of 293T cells with green fluorescence were observed. Western blotting confirmed that Porf-2 was successfully over-expressed. Conclusion  The co-expressing lentiviral vector of Porf-2 and GFP is successfully constructed; lentiviruses are packaged; and NG108, primary hippocampal neurons, and neural stem cells are transfected.

Key words: preoptic regulatory factor-2; , green fluorescent protein, lentivirus, neural cell