Construction of recombinant adeno-associated virus vector expressing the human tissue kallilrein

Objective To construct the recombinant adeno-associated virus vector expressing the human kallikrein and measure its titer.Methods A 786bp fragment cDNA containing human tissue kallikrein gene was successfully amplified by using PCR and directional cloned into pAAV-MCS plasmid.Recombinant adeno-associated virus AAV/HK was derived by co-transfecting DNAs of pAAV-HK and pAAV-RC、pAAV-Helper into AAV-293 cells using liposome methods and the replication-deficient HK recombinant adeno-associated virus was generated efficiently by homologous recombination.Meanwhile, pAAV-LacZ、 pAAV-RC and pHelper were co-transfected into AAV-293 cells also.The titer of recombinant AAV was calculated by means of staining the cells which expressed the report gene LacZ with X-Gal.Results The results of DNA sequencing showed that the fragment cloned in pAAV-MCS was HK-cDNA.The human kallikrein recombinant adeno-associated virus was obtained with the titers of 6.2× 107 particles/ml.Conclusion rAAV/HK can express human tissue kallikrein gene in HT-1080cells stably.The Success in constructing recombinant AAV/HK will promote the further research in molecular biology of human tissue kallikrein and the recombinant AAV/HK can be potentially used in cardiovascular diseases gene therapy.