中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (11): 1951-1954.doi: 10.3969/j.issn.1673-8225.2010.11.013

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

靶向人血管紧张素原小干扰RNA表达质粒的构建和鉴定

伍丽华，杨汝德   

  1. 华南理工大学生物科学与工程学院，广东省广州市 510640
  • 出版日期:2010-03-12 发布日期:2010-03-12
  • 通讯作者: 杨汝德，教授，华南理工大学生物科学与工程学院，广东省广州市 510640 rdyang@163.com
  • 作者简介:伍丽华，女，1984年生，江西省九江市人，汉族，华南理工大学生物科学与工程学院在读硕士，主要从事微生物制药方面的研究。 andyxian@21cn.com

Construction and identification of small interfering RNA expression plasmid target to angiotensinogen

Wu Li-hua, Yang Ru-de   

  1. College of Bioscience and Bioengineering, South China University of Technology, Guangzhou  510640, Guangdong Province, China
  • Online:2010-03-12 Published:2010-03-12
  • Contact: Yang Ru-de, Professor, College of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510640, Guangdong Province, China rdyang@163.com
  • About author:Wu Li-hua, Studying for master’s degree, College of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510640, Guangdong Province, China andyxian@21cn.com

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摘要：

背景：RNA干扰技术通过将具有一定结构特点、长19~25 bp的双链小干扰RNA导入哺乳动物细胞，特异性降解与其序列具有同源性的mRNA分子，导致目的基因表达抑制。

目的：拟构建针对人血管紧张素原mRNA的小干扰RNA表达载体，从而抑制肾素基因在脂肪细胞的表达。

方法：从NCBI中查找人血管紧张素原基因全长mRNA序列(NM000029)，利用GeneScript公司提供的在线小干扰RNA模板序列设计软件，自行设计靶向血管紧张素原的两条shRNA的DNA模板单链，合成靶向血管紧张素原基因转录可形成茎环结构的寡聚核苷酸，退火后与酶切后的psiRNAT-U6.1/Neo质粒连接，在TOP10菌株中扩增，并测序鉴定。

结果与结论：将含有血管紧张素原-mRNA目标序列19 bp的双链DNA插入片段，连接到pRNAT-U6.1/Neo质粒形成重组质粒。EcoRⅠ和Hind Ⅲ双酶切后，空载体得到351 bp小片段，而人重组质粒得到397 bp小片段，与预期相符。EcoRⅠ和Kpn Ⅰ双酶切后，空载体得到1条345 bp小片段，而人重组载体没有得到小片段条带，与预期相符。测序结果表明psiRNAT-U6.1/Neo质粒已经插入人脂肪细胞的干扰合成片段，无碱基突变，成功构建了靶向血管紧张素原-小干扰RNA表达载体。

关键词: 血管紧张素系统, 血管紧张素原, 血管紧张素转换酶, 小干扰RNA, 构建, 质粒

Abstract:

BACKGROUND: In mammalian cells, introduction of double-stranded small interfering RNA (19-25 bp) can cleave and destroy the cognate RNA, which can result in suppression of gene expression.
OBJECTIVE: To construct siRNA expression plasmid for interference angiotensinogen (AGT), thereby, to resist AGT expression in adipose cells.
METHODS: The mRNA sequence of AGT gene was searched from NCBI (NM000029). Utilize of GenScript siRNA technology, AGT-siRNA oligonucletides were chemically synthesized and inserted into pRNAT-U6.1/Neo vector after annealing, then transformed into TOP10. The recombinant plasmid was identified by restriction endonuclease and DNA sequencing.
RESULTS AND CONCLUSION: The recombinant plasmid psiRNAT-U6.1/Neo-AGT was obtained by connecting 19 bp segment containing AGT-mRNA sequence to pRNAT-U6.1/Neo. After EcoR Ⅰ and Hind Ⅲ digestion, 351 bp segment was obtained from empty vector, and 397 bp fragment band was obtained form recombinant plasmid, which was coincidence to the expectation. DNA sequencing showed Targeting siRNA oligonucleotides were correctly inserted into the eukaryotic expression vector pRNAT-U6.1/Neo without base mutation. The interference vector psiRNAT-U6.1/Neo-AGT was successfully constructed.

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