大鼠EAAT3真核表达载体的构建及在Hella细胞中的表达

Construction of eukaryotic expression vector carrying rat EAAT3 gene and its expression in Hella cells

目的:克隆大鼠谷氨酸转运蛋白3(EAAT3)的cDNA,构建其真核表达载体并转染Hella细胞.方法:从大鼠的脑组织中提取RNA,采用RT-PCR技术扩增EAAT3的基因编码区序列,将序列定向克隆至真核表达载体pcDNA3.1(+).经酶切及测序鉴定后用阳离子脂质体将其转染到Hel-la细胞中,通过免疫印迹法鉴定其表达.结果:经双酶切、测序鉴定证实EAAT3基因的真核表达载体构建成功.免疫印迹法证实EAAT3基因的表达.结论:成功克隆了EAAT3编码区序列,并构建了其真核表达载体,为以EAAT3为基础的中枢神经系统疾病的治疗奠定了基础.

AIM： To clone rat excitatory amino acid transporter 3 （EAAT3） cDNA and to obtain eukaryotic expression vector carrying EAAT3 gene and study the expression of EAAT3 gene in transfected Hella cell. METHODS： The cDNA of EAAT3 was amplified by RT-PCR. After purification, the gene fragment was cloned into a vector pcDNA3.1 （ ＋ ）. The sequence of inserted EAAT3 gene fragment was identified by enzyme digestion of EcoR I/Xho I and sequencing, and then the recombinant plasmid was transfected into COS7 cells using lipofectamine 2000. After 48 h, total protein was extracted and the expression of the EAAT3 gene in transfected Hella cells was identified by Western Blot. RESULTS： A fragment of 5.5 kb and inserted fragment of 1.5 kb were obtained by cutting positive recombinant plasmid of pc-DNA3.1 （ ＋ ）/EAAT3 with EcoR I/Xho I. Automatic DNA sequence analysis demonstrated that the sequence of the recombinant plasmid pcDNA3. 1 （ ＋ ）/EAAT3 was in accordance with that published in GenBank. The expression of EAAT3 gene was detected by Western Blot. CONCLUSION： The EAAT3 cDNA has been obtained and its recombinant eukaryotic expression vector has been successfully constructed. The study offers an important clue for the study and treatment of diseases from EAAT3 gene disorders in central nervous system.