逆转录法筛选mRNA靶点设计核酶对GPA的表达干预实验研究

doi:10.13523/j.cb.20140312

中国生物工程杂志

2014, Vol. 34

Issue (3): 84-90 DOI: 10.13523/j.cb.20140312

技术与方法

逆转录法筛选mRNA靶点设计核酶对GPA的表达干预实验研究

付辉^1,2, 李菲菲², 马琼², 付怀秀², 崔玉芳², 毛建平²

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

Experimental Screening mRNA Targets by Reverse Transcription for Ribozyme to GPA Expression Interference

FU Hui^1,2, LI Fei-fei², MA Qiong², FU Huai-xiu², CUI Yu-fang², MAO Jian-ping²

1. Medical University of Anhui, Hefei 230032, China;
2. Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China

全文: PDF(1473 KB) HTML

摘要：

采用自行设计5’固定3’随机的文库对基因mRNA进行杂交用于逆转录反应以筛选mRNA的寡核苷酸结合靶点。对人I型跨膜糖蛋白—血型糖蛋白A （glycophorin A，GPA）的mRNA筛选了4个反义寡核苷酸可结合靶点，分别设计反义核酸（Antisense），分别加入mRNA中用RNase H验证各靶点的核酸结合和切割效率，最终确定2个高效结合和切割靶点。再设计Ribozyme，构建表达核酶（Ribozyme）质粒，利用慢病毒（Lentivirus）包装技术，感染人源红系白血病细胞株K562细胞，在细胞水平验证其下调GPA基因表达的效果，对转染细胞mRNA进行反转录和Real Time PCR分析mRNA表达水平，并在蛋白水平进行了Western Blot分析。结果表明文库结合逆转录方法筛选靶点设计的Ribozyme具有高效率下调膜受体表达的作用，GPA为I型跨膜糖蛋白，该实验为筛选mRNA靶点提供参考方法，并对膜受体表达干预有参考价值。

关键词： 血型糖蛋白A; mRNA; 靶点; Ribozyme; 慢病毒; K562细胞

Abstract:

An oligodeoxynucleotide library which the sequence were defined at 5 prime and randomized at 3 prime was employed to screen mRNA accessible targets, in reverse transcription and PCR after hybridized the library with mRNA. The mRNA of Glycophorin A(GPA), type I transmembrane glycoprotein, was screened and obtained 4 targets sequences. Accordingly 4 antisense nucleic acids designed respectively, the binding efficiency of every target were verified by using RNase H with antisense nucleic acids. Among them 2 targets showed better effects on binding and cutting. Designed 2 ribozymes to these targets, packaged in lentivirus system, then infected K562 cells(human erythroid leukemia line), the down-regulation effect of gene expression was validated by Real Time RT-PCR and by Western Blot. It was found that the screened targets showed the best effective knocking down effects on gene expression. The study provided a reference for mRNA targets screening and Ribozyme design, and was helpful in membrane receptor expression interference, since GPA is a transmembrane protein.

Key words: Glycophorin A mRNA Target Ribozyme Lentivirus K562

收稿日期: 2014-01-21 出版日期: 2014-03-25

ZTFLH:

Q522

基金资助:

国家自然科学基金资助项目（81072565，30271546）

通讯作者: 毛建平 E-mail: maojp@nic.bmi.ac.cn

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引用本文:

付辉, 李菲菲, 马琼, 付怀秀, 崔玉芳, 毛建平. 逆转录法筛选mRNA靶点设计核酶对GPA的表达干预实验研究[J]. 中国生物工程杂志, 2014, 34(3): 84-90.

FU Hui, LI Fei-fei, MA Qiong, FU Huai-xiu, CUI Yu-fang, MAO Jian-ping. Experimental Screening mRNA Targets by Reverse Transcription for Ribozyme to GPA Expression Interference. China Biotechnology, 2014, 34(3): 84-90.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140312 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I3/84

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