Construct Eukaryotic Expression Vector PEGFP-N1-ALDH2 And It's Mutant Vector

Objective:To construct an eukaryotic expression vector pEGFP-N 1-ALDH2and it's mutant vector pEGFP-N1-ALDH2 (AAA) then transfect into PC 12 cells. Verify the fact that the PC 12 cells which was transfacted into EGFP-N1-ALDH2 will over-expr-ession ALDH2 while transfacted into mutant plasmid will repress ALDH2 activity. As the solid foundation for our later research of the protect of ALDH2 for PC 12 cells when it is experienced of hyoxia-reoxygenation.Methods:(1),Amplify ALDH2 gene by PCR and clone the DNA of ALDH2 gene into plasmid pUC57, Construct plasmid pUC57-simple-ALDH2 vector then transfec--ted into DH5a competent cell,extract positive clone and sequence pUC57-simple-ALDH2gene by double restriction enzyme digestion. (2),Clone pUC57-simple--ALDH2 vector into eukaryotic expression vector pEGFP-Nl,construct pEGFP-N1--ALDH2 eukaryotic expression vector. sequence pEGFP-N 1-ALDH2 gene by double restriction enzyme digestion. (3),construct mutant ALDH2 gene(Aldh2 2/2),codon Glu506 (GAA)was substituted with Lys (AAA) by sit-directed mutagenesis methods (4),transfect pEGFP-N1-ALDH2 and pEGFP-N1-ALDH2(AAA) into PC 12 cell via liposomes respectively.Result:(1),the ALDH2 gene have successfully amplified.(2)The clone vector pUC57-Simple-ALDH2 was successfully constructed.(3),The eukaryotic expression vector pEGFP-N1-ALDH2 was successfully constructed.(4),The eukaryotic expression vec-tor pEGFP-N1-ALDH2 (AAA) was successfully constructed. (5),transfect pEGFP-N 1-ALDH2 and pEGFP-N 1-ALDH2 (AAA) into PC 12 cell successfullyConclusion:(1). The ALDH2 gene have successfully amplified by PCR. Electro-phoresis manifest have gain right DNA strap.(2).Clone ALDH2 gene into pUC57, sequence have no mismatching base pair then clone pUC57-ALDH2 into pEGFP -N1,construct pEGFP-N1-ALDH2 eukaryotic expression vector,both electrophoresis and DNA sequence have manifested right.(3). ALDH2 gene codon Glu506 (GAA)was substituted with Lys (AAA) by sit-directed mutagenesis methods,gain mutant ALDH2 gene(Aldh2 2/2)and construct pEGFP-N1-ALDH2 (AAA) vector successfully. (5),two vectors were transfected into PC 12 cells,screen out stabilization cell line. pEGFP-Nl-ALDH2 cell line expression ALDH2 protein,while pEGFP-N1-ALDH2 (AAA) cell line does not expression.