miR-15a/16-1慢病毒表达载体的构建及鉴定

张春鸿; 方潇碧; 张露; 王珍珍; 武鹏; 廖志苏

doi:10.13201/j.issn.1001-1781.2014.12.016

miR-15a/16-1慢病毒表达载体的构建及鉴定

- 温州医学院附属第一医院耳鼻咽喉科(浙江温州, 325000)
基金项目:
浙江省科技计划项目基金资助（No：2010C34005）

详细信息

通讯作者: 廖志苏,E-mail:liaozhisu111@163.com

中图分类号: R762

收稿日期: 2013-12-22

Construction and identification of lentiviriral vector over-expressing miR-15a/16-1

- Department of Otolaryngology, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, China

More Information

Corresponding author: LIAO Zhisu, E-mail: liaozhisu111@163.com

Received Date: 22 December 2013

摘要

摘要: 目的：构建miR-15a/16-1过表达慢病毒载体，并对其进行病毒包装、鉴定、滴度测定与感染靶细胞。方法：利用PCR法钓取miR-15a/16-1序列片段；将目的基因miR-15a/16-1克隆到慢病毒载体LV3中，经双酶切及测序鉴定并大量抽提；利用脂质体将重组质粒和包装质粒pGag/Pol、pRev、pVSV-G共转染293T细胞包装产生慢病毒。收集病毒悬液梯度稀释，感染293T细胞进行病毒滴度检测；将所得慢病毒感染靶细胞。结果：酶切与测序结果证明成功构建重组质粒，并成功包装成慢病毒，实验组病毒滴度为1×10⁹TU/ml，对照组病毒滴度为2×10⁹TU/ml。慢病毒成功感染靶细胞，其转染效率可达到80%左右。结论：成功构建miR-15a/16-1慢病毒表达载体，可获得高效miR-15a/16-1的慢病毒颗粒。
- miR-15a/16-1 /
- 慢病毒 /
- 293T细胞 /
- 绿色荧光蛋白
Abstract: Objective: To construct a lentiviral vector that over-expression miR-15a/16-1, and to proceed with the lentivirus package, identification, titer determination and transfection to target cells. Method: miR-15a/16-1 sequence was obtained by PCR, and it was inserted into the lentiviral vector LV3, and then it was identified by double-enzyme cleavage and gene sequence analysis and extracted. The recombinant plasmid and packaging plasmid pGag/Pol, pRev, and pVSV-G were co-transfected into 293T cells with the application of liposomes to package the lentivirus. The viral suspension was collected and diluted with gradient, then it was transfected to 293T cells and the titre was determined, and last obtained lentivirus was transfected to target cells.Result: Restriction enzyme digestion and sequencing results showed that recombinant plasmid has been successfully constructed and packaged to lentivirus. The viral titer was 1×10⁹ TU/ml in experimental group, and 2×10⁹ TU/ml in control group.The target cells were successfully transfected and the transfection efficiency could reach to 80%. Conclusion: It was successful to construct lentiviral vector of miR-15a/16-1 and to obtain efficient lentiviral particle of miR-15a/16-1.
- miR-15a/16-1 /
- lentivirus /
- 293T cells /
- green fluorescent protein

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