Preparation Of DNA Aptamers Against Recombinant Adeno-associated Virus And Their Trial Application In Purification Of RAAV

The preparation of recombinant adeno-associated virus(rAAV)as a vector for development of vaccine and gene therapy has attracted the attention of the industry,but its mass production has many shortcomings,especially the high difficulty of purification,the limitation of serotype dependence,the inability of large-scale preparation by gradient centrifugation,the high cost of mixed chromatography and the cumbersome steps of molecular exclusion purification.The progress of rAAV purification process depends on the emergence of new affinity materials.Based on the combination of heparin and heparin sulfate receptor of type 2 AAV(AAV2)capsid,this study improved Systematic Evolution of Ligands by Exponential Enrichment(SELEX)technology,fixed rAAV with heparin beads,involed inactivated virus for negative seletion in rounds 6-10,and carried out dot blotting assay in round11 library.After the positive characterization results of Eastern blotting,T5 zero plasmid was introduced to screen positive recombinants.A group of DNA aptamer sequences specifically binding to rAAV-EGFP have been screened,which have not been reported.The selected aptamers were characterized by dot blot,Eastern blotting and QCM-D tests;The color changes of nano-gold particles has a good linear relationship between 0.4vg/m L and 1vg/m L;The Kd value determined by ELISA was20.71 nmol/m L.The obtained aptamers were used as affinity material to further study the purification of rAAV from cell lysate.In this study,the aptamers with affinity of broadspectrum rAAV serotype were coated on the surface of magnetic beads for their functionalization.After optimizing its functionalization conditions,a new affinity material that can selectively bind to recombinant adeno-associated virus(rAAV)was chosen.The functionalized magnetic beads were used to extract and purify adenoassociated virus vector particles from host cell lysates.The isolation of purified virus can be separated directly using magnetic rod or magnetic frame.Aptamers well combined with streptavidin magnetic beads to prepare affinity column for rAAV purification.After purification,rAAV cell lysate was subjected to protein electrophoresis,and 60 k Da: 72 k Da: 90 k Da = 10:1:1.The purification recovery was optimized.The optimal proportion of streptavidin magnetic beads and biotin modified SEQ ID: 23,the incubation time of streptavidin magnetic beads and biotin modified aptamer,the loading of rAAV by aptamer functionalized magnetic beads,and the streptavidin magnetic beads in the purification system were optimized by orthogonal experiment.SEQ ID No: 23,The optimal elution temperature,elution buffer and sample loading volume of the purification system were determined by orthogonal experiment to verify the experimental repeatability of the optimized conditions.The rAAV purified by aptamer functionalized magnetic beads was removed by Q-type strong anion exchange column,and the AAV after removing the empty shell was transfected into He La cells by strong anion exchange column.The transfection efficiency was judged by the number of positive cells.In the control experiment,aptamer functionalized magnetic beads can be used to purify rAAV-5-GFP and recombinant adeno-associated virus rAAV-7-GFP that cannot be purified by heparin.Carboxyl magnetic beads are coupled with AAV antibody to prepare antibody immunomagnetic beads as control.The purification capacity of aptamer functionalized magnetic beads is compatible with that of antibody.However,aptamer-based process is simpler with lower cost.