人tRNA^(ser)基因真核表达质粒的构建及对FRL-19肝癌细胞的高效转染

Construction of recombinant vector expressing human tRNA ^((ser)(CGA)) and optimal transfection in FRL-19cells

目的:构建人tRNAser(CGA)基因与真核表达载体pSV2neo的重组体,探讨细胞高效转染新方法,研究tRNA种类及丰度对转录后翻译水平的影响;为探索基因治疗新思路打下基础。方法:EcoRI酶切pGEM-TeasytRNAser(CGA)质粒释放目的基因tRNAser(CGA)片断,与pSV2neo载体构成重组体,双酶消化鉴定重组载体插入方向。扩增纯化构建的pSV2neotRNAser(CGA)质粒,经脂质体和Plus试剂混合包装,转染FRL-19肝癌细胞株。用Hirt方法提取胞浆DNA,做Southernblot。结果:用Nsil和BamHI双酶切鉴定获得正确方向的重组质粒pSV2neotRNAser(CGA),DNA测序显示其序列正确。Southernblot结果显示:在400bp处可见一条特异DNA条带,说明pSV2neotRNAser(CGA)已转入FRL-19细胞。结论:成功构建人pSV2neotRNAser(CGA)真核表达质粒。Plus试剂可明显提高脂质体转染FRL-19细胞的效率。

Objective:The re combinant vector expressing human tRNA ser(CGA) was constructed to in-vestigate whether the alteration of the tRNA profiles alters the transla tion efficiency of a given gene and increase the transfection activity in FRL-19liver cancer cell line.Methods:The tRNA ser(CGA) DNA frag ment was di gested by EcoRI from pGEM-T easy tRNA ser(CGA) and in serted into eukaryotic vector pSV2neo.The re-com binant vector was transfected into FRL-19cell by lipofectMINE and Plus reagents.The Hirt DNA was extracted,and analysed by Southern blot.Results:The re combinant vector pSV2neo tRNA ser(CGA) was suc-cessfully constructed according to the DNA se quencing and enzyme digestions.The specific tR NA ser(CGA) DNA fragment was ob tained from the transfected cell by Southern blot analysis.Plus reagent can shorten the transfection time and decrease the quantities of plasmid DNA.Conclusion:The recombi nant eukaryotic vec-tor pSV2neotRNA ser(CGA) was successfully constructed and transfected into the FRL-19cell line.The lipofectamine plus reagent en hance the transfection activity in the cell.The pre sent work is the basis for futher research on the gene therapy.