microRNA过表达载体构建技术的实验研究

Practical study on technology of construction of microRNA overexpression vector

目的:构建microRNA的表达载体,再转染生物体细胞观察该microRNA的转录或表达情况,以获知它的作用机理及对生物体的影响。方法:以microRNA-21(简化为miR-21)为例说明构建表达载体的方法。根据microRNA的成熟序列以及附近约200多碱基共约470个碱基序列,设计PCR引物,PCR扩增,PCR产物和pcDNA3.1(+)质粒双酶切后,用T4连接酶进行连接反应,并转化入感受态细胞,筛选后对重组质粒进行双酶切、琼脂糖凝胶电泳鉴定及测序分析,并将其转染Hela细胞,经RT-PCR实验检测其表达情况。结果:pcDNA3.1(+)/miR-21过表达载体转染细胞后使得miR-21在细胞中过表达。结论:成功构建了miR-21的真核表达载体,为进一步研究miR-21功能及作用机制奠定实验基础。

This article introduces how to construct miRNA-21 overexpression vector and transfect it into cell （ Hela）. Then the level of expression of miR-21 was observed. The miR-21 precursor is amplified by PCR method ,then the recombinant vector pcDNA3.1 （＋ ） and miR-21 are constructed. After screening and identifying the recombinant ,it is transfected into cancer cells （Hela）.The miR-21 expressing level is detected by RT-PCR.The result shows that the level of the miR-21 expression increases significantly in the cells that are transfected the recombinant. The miR-21 overexpression vector is successfully constructed ,and a foundation for studying the function of miR-21 is established .