miR-610的克隆及其慢病毒表达载体的构建

Cloning of has-miR-610 and Construction of Its Lentiviral Expression Vector

目的:克隆微小RNA hsa-miR-610并构建其慢病毒表达载体。方法:将PCR扩增得到的miR-610前体序列和pcDNA6.2-GW/EmGFP载体经双酶切后连接产生pcDNA6.2-miR-610真核表达载体,双酶切后测序鉴定,筛选阳性克隆。通过BP反应及LR反应将pre-miR-610克隆至慢病毒目的载体pLenti6/V5-DEST,构建miR-610的慢病毒表达载体pLenti6/V5-miR-610。结果:将构建好的慢病毒表达载体导入大肠杆菌Stbl3进行扩增,测序表明所构建的载体与预期的完全一致。结论:成功构建了miR-610的慢病毒表达载体pLenti6/V5-miR-610,为深入研究miR-610的生物学功能奠定了基础。

Objective： To construct a lentiviral expression vector for hsa-miR-610.Methods： The pre-miR-610 amplified by PCR was inserted into pcDNA6.2-GW/EmGFP.The recombinant plasmid pcDNA6.2-miR-610 was confirmed by restriction endonuclease analysis.Using Gateway cloning technology,the lentiviral expression vector miR-610 was constructed by BP and LR recombination reactions.The recombinant plasmid pLenti6/V5-miR-610 was confirmed by DNA sequencing.Results： DNA sequencing demonstrated that the lentiviral expression vector pLenti6/V5-miR-610 was constructed successfully.Conclusion： The successful construction of lentivirus vector pLenti6/V5-miR-610 provides the basis for further study of molecular functions of miR-610.