Construction Of Recombinant Adeno-Associated Virus Vector Expressing Human Tissue Kallikrein Gene And Delivery In Human Umbilical Vein Endothelial Cells

Objective To construct the recombinant adeno-associated virus vector expressing human tissue kallikrein gene and detect the expression of interested gene in umbilical vein endothelial cells(HUVEC) which were infected with different titer of recombinant AAV. Discuss the feasibility of gene therapy for hypertension by recombinant adeno-associated virus vector which carry human tissue kallikrein gene.Methods 1. The KK gene was directional cloned into the pAAV-MCS. The recombinant pAAV expression plasmid was called pAAV/KK. 2. Then co-transfect AAV-293 cells with three plasmids(pAAV/KK, pAAV-RC, and pHelper) by Iipofectamine2000. Meanwhile, pAAV-LacZ control plasmid was co-transfected together with pAAV-RC, and pHelper into the AAV-293 cells in parallel with the recombinant plasmid containing the KK gene in the same transfectional condition. The AAV-293 cells containing pAAV-LacZ-derived virus can be stained by In Situ Galactosidase Staining Kit (X-gal) to confirm that the AAV-293 transfection was successful. Harvest the recombinant AAV particles containing LacZ. Dilute viral stocks over a 10-fold series from 10-2 to 10-5 and infect HT-1080 cells with different folds of diluted viral stocks. HT-1080 cells containing pAAV-LacZ-derived virus was stained by In Situ Galactosidase Staining Kit to estimate the viral titer of harvested AAV particles. 3. Infect HUVEC cells with different titer of AAV particles. 72 hours later, detect the expression of human tissue kallikrein gene by semi-quantitave RT-PCR.Results 1. Successfully construct the recombinant AAV expressing system of human tissue kallikrein gene. 2. The viral titer of recombinant AAV measured by staining of In Situ Galactosidase Staining Kit is 6.2 107 particles/ml. 3. WhenHUVEC cells was infected with the recombinant AAV particles contain kallikrein gene at different liters, in contrast with the blank group, the expression of human tissue kallikrein gene increased significantly in the group of 1X 10~6 1 X 105 1 X 10~4 (p<0.05), especially in the group of 1 X 10~6(P<0.001). But there was no significant increase in the group of 1 X 103 when compared with the blank group (P>0.05).Conclusion When co-transfecting AAV-293cells with three plasmids(the recombinant pAAV expression plasmid, pAAV-RC, and pHelper), the liter of recombinant AAV particles can reach > 107 particle/ml stably. The gene of interested can be expressed significantly and stably when using recombinant AAV viral to infect the cultured mammal cells. This study has established a foundation in gene therapy for hypertension in practice.