Package and titer assay of recombination adeno-associated virus with c-Met short hairpin / 吉林大学学报(医学版)

ABSTRACT

Objective To package the recombination adeno-associated virus with human U6 promoter and c-met short hairpin in order to establish foundation for research on inhibiting the expression of c-Met and cancer gene therapy.Methods c-Met short hairpin was synthesized and linked to the down stream of U6 promoter by PCR.Adeno-associated viral vectors pSNAVUshMet(1,2) were constructed and transfected into BHK cells.Positive cell clones were selected by G418.Adeno-associated virus with U6shMet were packaged by adding rHSV1-repcap.The virus purity was analysed by SDS-PAGE and titer was assayed by dot blot.Results Two fragments(U6shMet1 and U6shMet2) with c-Met short hairpin were obtained and two recombination adeno-associated virus(rAAVUshMet1 and rAAVUshMet2) with U6shMet were packaged successfully.SDS-PAGE analysis showed that the purified virus appeared clear characterized bands.The virus titer was 4?1012mg?L-1.Conclusion The recombination adeno-associated virus(rAAVUshMet1 and rAAVUshMet2) with U6shMet are constructed successfully.The virus have high titer and good purity and could act as the effective vehicle of inhibiting the expression of c-Met and cancer gene therapy.