Establishment Of Cell Model Over-expression Rat ACE And Study On Preliminary Screening Of ACE Inhibitors

Objectives Construct the ACE gene from the lung of rat eukaryotic expression vector pEGFP-ACE,and detect its expression in HepG2 cell for the establishment of cell model expressed ACE and to screening drug on ACEI.Methods 1. The total RNA of the rat lung was extracted using Trizol reagent. The complete encoded sequence of the rat ACE gene was amplified by RT-PCR from rat lung. The amplified target gene was inserted into eukaryotic expression vector pEGFP-C1 to construct the recombined plasmid that encoded ACE cDNA.2. HepG2 cells was transfected by electroporation by 250v/20ms, the expression protein was detected by Western Blot after 48h and then selected the over-expression rat ACE by G418.3. ACE can catalyst HHL decomposition of HA ,then using HPLC detemination HA of the reaction solution to detect the avtivity of ACE;using captopril to detect the cell model .Results 1. ACE cDNA from rat lung was successful obtained by RT-PCR.We successful obtained the recombinant plasmid pEGFP-ACE through confirmation by digestion with SalI and Acc65I and by sequencing and Nucleotide homology analysis to GeneBank.2. The recombinant plasmid transfected HepG2 cells by electroporation 48 hours after transfection, the recombined ACE protein about 180KD was detected.3.The cell model of over-expression ACE was comfirmed by HPLC on catalytic HHL significant difference compared to HepG2 cell;the captopril can specific inhibition ACE on this cell model.Conclusions We successful construct the eukaryotic expression vector of rat ACE gene from rat lung;after transfecting HepG2 cells, the recombinant ACE protein is detected,then we get the cell model of over-expression ACE; the captopril can specific inhibition ACE on this cell model confirm which can be used provide basis for drug screening on ACEI.