pRNA导入型Ribozyme的设计和体外转录制备

中国生物工程杂志

2011, Vol. 31

Issue (06): 99-105

技术与方法

pRNA导入型Ribozyme的设计和体外转录制备

周颖^1,2, 方静², 毛建平², 祝延¹

1. 安徽医科大学基础医学院合肥 230032;
2. 军事医学科学院放射与辐射医学研究所北京 100850

The Design and in vitro Transcription of pRNA-Ribozymes

ZHOU Ying^1,2, FANG Jing², MAO Jian-ping², ZHU Yan¹

1. Basic Medicine College, Anhui Medical University, Hefei 230032, China;
2. Beijing Institute of Radiation Medicine, Beijing 100850, China

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摘要：

目的:枯草杆菌的包装RNA分子pRNA是新型纳米分子载体,将其同锤头型核酶Ribozyme重组可以构建结构稳定、能进入细胞、主动识别结合和剪切基因RNA的pRNA-Ribozyme。由于目前100 nt以上的RNA分子采用化学合成制备较为困难,实验采用基因重组构建并体外转录制备170 nt的pRNA-Ribozyme。方法:依据pRNA序列和Ribozyme分子序列,采用PCR扩增和克隆重组,将pRNA-Ribozyme按照顺式连接开放式结构、和Ribozyme嵌入pRNA成自身环化闭合型结构,制备其DNA质粒;再在体外转录制备pRNA-Ribozyme分子。结果:体外生物合成制备pRNA-Ribozyme顺式连接开放式结构、和Ribozyme嵌入pRNA成自身环化闭合型结构pRNA-Ribozyme分子,它们能通过自身正确折叠形成聚合体结构。体外鉴定pRNA-Ribozyme对其靶mRNA均具有切割降解活性,为pRNA-Ribozyme分子的细胞内应用和在胞内表达应用打下基础。

关键词： pRNA; Ribozyme

Abstract:

Objectives: pRNA, the packaging-RNA molecule of Bacillus subtilis is a new nanometer-molecular carrier. The recombination of pRNA and the hammerhead Ribozyme can be used to construct stable, self-deliverable pRNA-Ribozymes for entering cells and actively binding and cutting RNA. However, it is technically difficult to synthesize RNA molecules longer than 100 nt by chemistry. Therefore, 170 nt pRNA-Ribozyme was prepared by genetic recombination and in vitro transcription. Methods: The DNA of pRNA and Ribozyme were amplified by PCR before recombination according to their molecule sequences. The pRNA-Ribozyme was reconstructed in two forms: the chimeric ribozyme linked to pRNA vector as a cis-connected open structure, and the chimeric ribozyme harbored in pRNA vector as a closing inner structure. The pRNA-Ribozymes were obtained by in vitro transcription. Results: The pRNA-Ribozyme of cis-connected open structure and the embedded inner structure were prepared by in vitro transcription. Both molecules could form the polymer structure in vitro through correct folding. The pRNA-Ribozyme had the activity for binding and cutting its target mRNAs, which provides the foundation for further application of pRNA-Ribozyme in intracellular.

Key words: pRNA Ribozyme

收稿日期: 2011-02-25 出版日期: 2011-06-28

ZTFLH:

Q819

基金资助:

科技重大专项(2008ZXJ09001-014)资助项目

通讯作者: 毛建平 E-mail: maojp@nic.bmi.ac.cn; zhuyan@ah.edu.cn

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引用本文:

周颖, 方静, 毛建平, 祝延. pRNA导入型Ribozyme的设计和体外转录制备[J]. 中国生物工程杂志, 2011, 31(06): 99-105.

ZHOU Ying, FANG Jing, MAO Jian-ping, ZHU Yan. The Design and in vitro Transcription of pRNA-Ribozymes. China Biotechnology, 2011, 31(06): 99-105.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I06/99

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