Hath1基因真核表达载体的构建及其在SH-SY5Y细胞中的表达

Construction of eukaryotic expression vector of Hath1 gene and its expression in SH-SY5Y cells

目的:构建携带Hath1基因的真核表达载体并转染神经母细胞瘤细胞(SH-SY5Y),观察其在SH-SY5Y细胞中的表达。方法:从人全血中提取DNA,克隆Hath1基因,同时扩增绿色荧光蛋白(GFP)gfp基因;利用引物GFP-R和Hath1-F扩增融合基因gfp-Hath1,克隆至pMD18-T中;经XhoⅠ和EcoRⅠ限制性内切酶酶切,构建重组表达质粒pCDNA3.1(+)::gfp-Hath1;转染SH-SY5Y细胞。结果:PCR扩增,融合基因gfp-Hath1扩增条带大小为2 023bp,表明成功扩增gfp-Hath1融合基因。经双酶切鉴定,成功构建真核表达质粒pCDNA3.1(+)::gfp-Hath1。采用间接免疫荧光技术在荧光显微镜下观察到5H-SY5Y细胞中有GFP表达。结论:本研究成功地使Hath1基因在SH-SY5Y细胞中得到表达,为转染耳蜗组织的实验及探索治疗耳聋新方法奠定基础。

Objective To construct the eukaryotic expression vector of Hath1gene and to transfect neuroblastoma cells（SH-SY5Y）,and to observe its expression in SH-SY5Ycells.Methods DNA was extracted from human whole blood,the Hath1gene was cloned and the green fluorescent protein（GFP）gfp gene was amplified at the same time;the primers GFP-R and Hath1-F was used to amplify fusion gene gfp-Hath1,and gfp-hath1was cloned into pMD18-T and digested with XhoⅠ and EcoRⅠ.The recombinant expression plasmid pCDNA3.1（＋）：：gfpHath1was constructed and transfected into SH-SY5Ycells.Results The gfp-Hath1fusion gene with 2 023bp was successfully amplified.The eukaryotic expression plasmid pCDNA3.1（＋） ：：gfp-Hath1 was constructed successfully after identified with double enzyme digestion.The GFP was found in SH-SY5Y cells under immunofluorescence microscope.Conclusion The Hath1gene is expressed in SH-SY5Ycells successfully,which lays a foundation for transfection cochlea experiment and exploring new methods to treat deafness.