Construction of a Series of Adeno associated Virus Vectors and Their Expression of β-galactosidase Gene

In order to facilitate gene therapy research using recombinant adeno associated virus(rAAV) vector as gene transfer vehicle, we constructed a series of rAAV vectors including pWAV 1, pWAV 2 and pSNAV for inserting foreign genes, each of which is composed of the two ITRs of AAV 2 and in between are the human cytomegalovirus (CMV) immmediate early enhancer and promoter, multiple cloning sites(MCS) and polyadenylation signal. A rAAV vector carrying a gene expression cassete can be conveniently obtained by inserting the target gene into the MCS in a correct direction. The MCS of pWAV 1 includes NheⅠ,XhoⅠ,PstⅠand BamHⅠ, where up to 3.3kb foreign genes can be inserted for packaging of rAAV particles. Both pWAV 2 and pSNAV have the same capacity of inserting 3.6kb foreign genes, with MCS of KpnⅠ, EcoRⅠ, SalⅠ for the former and KpnⅠ, EcoRⅠ, SalⅠ and BglⅡ for the latter. We also constructed rAAV vectors pAV lacZ and pSNAV lacZ, both harboring β galactosidase gene. After transfecting pAV lacZ and pSNAV lacZ into BHK 21 cells respectively, a recombinant herpes simplex virus(rHSV) having the whole helper functions for rAAV packaging that we reported recently was used to infect the cells. Two strains of rAAV carrying β galactosidase gene were obtained with a titer of 5×10 4 TU/ml respectively, which not only confirmed the feasibility of this method but also indicated the possibility to produce rAAV in large scale using “one helper virus one vector cell line” strategy.