吉林大学学报(医学版)

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Hath1基因真核表达载体的构建及其在SH-SY5Y细胞中的表达

宋丽华1,齐 力1,王 敏1,迟玉涛1,许小敏2   

  1. 1.内蒙古民族大学附属医院耳鼻喉科,内蒙古 通辽 028000;
    2.内蒙古自治区赤峰市医院神经内科,内蒙古 赤峰024000
  • 收稿日期:2013-01-25 出版日期:2013-07-28 发布日期:2013-08-17
  • 作者简介:宋丽华(1972-),女,内蒙古自治区通辽市人,副主任医师,医学硕士, 主要从事耳鼻喉科的基础和临床研究。
  • 基金资助:

    国家自然科学基金资助课题(81160551/H2818);内蒙古自治区高等学校科学
    研究项目资助课题(NJZY13162)

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

SONG Li-hua1,QI Li1,WANG Min1,CHI Yu-tao1,XU Xiao-min2    

  1. 1.Department of Otorhinolaryngology,Affiliated Hospital,Inner Mongolia University for Nationalities,Tongliao 028000,China;2.Department of Neurology,Chifeng City  Hospital,Chifeng  024000,China
  • Received:2013-01-25 Online:2013-07-28 Published:2013-08-17

摘要:

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Xho Ⅰ和EcoR Ⅰ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2 023 bp,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。

关键词: Hath1基因, gfp基因, 真核表达, 荧光显微镜

Abstract:

Objective To construct the eukaryotic expression vector of Hath1 gene and to transfect neuroblastoma cells (SH-SY5Y),and to observe its expression in SH-SY5Y cells.Methods DNA was extracted from human whole blood,the Hath1 gene 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-hath1 was cloned into pMD18-T and digested with XhoⅠ and EcoRⅠ. The recombinant expression plasmid pCDNA3.1 (+) :: gfp-Hath1 was constructed and   transfected into SH-SY5Y cells.Results The gfp-Hath1 fusion gene with 2 023 bp 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 Hath1 gene is expressed in SH-SY5Y cells successfully, which lays  a foundation for transfection cochlea experiment and exploring new methods to treat deafness.

Key words: Hath1 gene, gfp gene, eukaryotic expression, fluorescence microscope

中图分类号: 

  • Q782