Construction And Identification Of Vector Carrying Rat Excitatory Amino Acid Transporter (EAAT) Gene

PartⅠConstruct and identify pAd/EAAT2Objective: To clone excitatory amino acid transporter2 (EAAT2) cDNA and construct pAd/EAAT2. Methods: The cDNA of EAAT2 were obtained by inverse transcription RNA of rat brain tissue , and inserted into the adenoviral shuttle vector–pTracer-GFP. Then it was linearized with PmeI and cotransformed with E1-deleted adenoviral backbone AdEasy-1 into the competent bacterial strain BJ5183, which allows efficient recombination to occur. After screening, recombinants for adenoviruses Ad/EAAT2 and Ad-GFP, which contains no insert sequence as a control, were generated. The recombinant DNA was identified by restriction endonuclease PacI and transfected into HEK 293 cells after linearization. The cells were collected when a cytopathic effect appeared, and the generated recombinant adenoviruses were isolated. The titers were determined by plaque assay of the infected HEK 293 cells with serially diluted supernatant. Results: The segment of EAAT2 was inserted into adenoviruse vector with GFP .The titers were 2×108 pfu/ml. Conclusion: pAd/EAAT2 is constructed successfully,which lays a basis for treatment and study of central nervous system diseases with lack of EAAT2.PartⅡConstruction of eukaryotic expression vector carrying rat EAAT3 gene and its expression in Hella cellsObjective :To clone rat excitatory amino acid transporter3 (EAAT3) cDNA and to obtain eukaryotic expression vector carrying EAAT3 gene and study the expression of EAAT3 gene in transfected Hella cel1. 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 EcoRl/Xhol and sequencing,and then the recombinant plasmid was transfected into Hella cells using lipofectamine 2000.After 48 h,total protein was extracted and the expression ofthe EAAT3 gene in transfected Hella cells was identified by Western Blot. Results: A fragment of 5.5kb and inserted fragment of 1.5kb were obtained by cutting positive recombinant plasmid of pcDNA3.1(+)/EAAT3 with EcoRI/Xhol. 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.