Establishment And Application Of Receptor-binding Capture Method Of Adeno-associated Virus

Adeno-associated virus(AAV)vectors have promising applications in gene therapy and vaccine development,but their use is limited by scale production and complicated purification procedure.Receptor-binding capture and magnetic sequestration has been used for concentration and survaillence viruses in environmental samples,which requires expensive equipment and reagents.Elastin-like polypeptide(ELP)has the temperature-sensitive reversible phase transition property and their fusion proteins can be purified simply by inverse transition cycling(ITC).AAV receptor(AAVR)contains 5 polycystic kidney domains(PKD),among which PKD1 and PKD2 are requied for AAV5 and AAV2 binding.In this study,both PKD1 and PKD2 were expressed in E.coli as an ELP fusion protein,aiming at establishing a simple method for AAV concentration and purification.PKD 1 and PKD2 gene segments of AAVR were cloned into ELP fusion expression vector pET-ELP and the recombinant vector pELP-PKD was transformed into E.coli strain BL21(DE3).The expression of ELP-PKD fusion protein was induced with 0.2mM IPTG for 18h at 20?.The transition temperature of ELP-PKD fusion protein and NaCl concentration for ITC purification were optimized.The results showed that an expected 73-kDa ELP-PKD fusion protein was expressed as a soluble protein.The temperature for ELP-PKD phase transition was 26? in the presence of 2M NaCl.ELP-PKD fusion protein was purified to 92%purity.Sf9 cells were co-transfected with pBacFast-RC and pBacFast-GFP and the resultant recombinant AAV was called AAV-GFP.AAV-GFP was incubated with an equal volume of ELP-PKD and the protein-virus complex was precipitated by ITC.The viral DNA was extracted and amplified by PCR.The conditions for AAV-GFP binding to and elution off ELP-PKD were optimized.The purified AAV-GFP was analyzed by electron microscopy,PK-15 cell infection,SDS-PAGE and Western blotting.The results showed that ELP-PKD fusion protein had a specific binding affinity for AAV-GFP.The optimal conditions for AAV-GFP binding were 100?g/mL of ELP-PKD for 15min at pH7.0 at 4?.The optimal conditions for elution of AAV-GFP was 30min at pH3.0 at 25?.The purified AAV-GFP could infect PK-15 cells with the typical AAV morphology and VP1:VP2:VP3 ratio of about 1:1:10.To obtain the cell lysates for AAV-GFP purification,pBacFast-RC and pBacFast-GFP vectors were co-transfected into insect cells,and pAAV-RC and pAAV-GFP vectors were co-transfected into AAV-293 cells.AAV-GFP was purified from the cell lysates by ELP-PKD binding capture and titrated on PK-15 cells before and after elution.The results showed that purification of AAV-GFP had a recovery rate of 44%from AAV-293 cells or 47%from insect cells.The purification could be complished within 1.6h.