人微管蛋白辅助因子A基因shRNA表达质粒的构建及鉴定

逄海港; 张鹏; 宋尔霖; 李新涛; 马鑫; 张旭

doi:10.13201/j.issn.1001-1420.2014.06.027

人微管蛋白辅助因子A基因shRNA表达质粒的构建及鉴定

- 解放军总医院泌尿外科(北京, 100853)
基金项目:
卫生行业科研专项项目:泌尿系统重大疾病的防治研究 (编号201002010)

国家重点基础研究发展计划 (973计划) (编号2013CB530803)

详细信息

通讯作者: 张旭,E-mail:xzhang@foxmail.com

中图分类号: R737.11

收稿日期: 2013-10-28

Establishment and identification of shRNA expressing plasmid targeting tubulin cofactor A gene

- Department of Urology, General hospital of PLA, Beijing, 100853, China

More Information

Corresponding author: ZHANG Xu, E-mail: xzhang@foxmail.com

Received Date: 28 October 2013

摘要

摘要: 目的:构建针对微管蛋白辅助因子A(TBCA)基因的shRNA载体并进行鉴定。方法:针对人TBCA基因mRNA序列,设计合成编码shRNA的两条寡核苷酸链,经退火形成发卡寡核苷酸模板片段,经双酶切克隆至pSilencer2.0-U6-neo和pGCsi-U6-neo-GFP载体,进行酶切测序鉴定,脂质体转染后进行western blot测定。结果:酶切测序证实成功构建干扰质粒,脂质体转染786-0细胞系后24小时可见绿色荧光蛋白。Western blot证实TBCA表达量降低。结论:成功构建了针对TBCA基因shRNA表达载体,为下一步进行RNAi的相关研究奠定了基础。
- 微管蛋白辅助因子A /
- shRNA /
- 基因沉默
Abstract: Objective:To construct the expressing vector encoding small hairpin RNA (shRNA) targeting tubulin cofactor A (TBCA) gene and to identify it.Method:According to the mRNA sequence of TBCA gene, two expression sequences of shRNA were designed and inserted into expression vector pSilencer2.0-U6-neo and pGCsi-U6-neo-GFP. The recombined plasmid vectors were identified by restriction enzyme analysis and sequencing. After transfecting into 786-0 cell, western blot was used to identify the expression of TBCA.Result:The interferential plasmid was constructed correctly, which was confirmed by restriction endonuclease digestion and sequencing analysis. After transfecting 786-0 cell lines, green fluorescent protein was found. Western blot indicated that the expression of TBCA decreased.Conclusion:The interferential plasmid was constructed successfully so as to lay a foundation for the study of RNAi.
- TBCA /
- short hairpin RNA /
- gene silencing

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参考文献(8)

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