Preparation Of VP2、VP3and AAP For AAV Capsid In Vitro Self-assembly

Objective: The development of AAV vector in gene therapy has been into thephase of clinical trials. But the single batch production of vector particles is requiredup to1015VP or even more for general big animals’ pre clinical studies orexperiments and this results in a great challenge for current in vivo virus preparation,then it also increase the prime cost. Therefore, we have intended to make rAAVartificially: i.e. First of all, we prepared individual AAV protien elements and DNAfor AAV assembly through genetic engineering methods. Second, the AAV-VLPwould be self-assembled in vitro by nanometer technology. Finally it would becomeactive virus after the genome’s encapsidation. The aim of this study is to preparecapsid proteins and AAP.respectively.Methods: pichia pastoris was used for construction of engineering strainsGS115-VP2and GS115-VP3. The proteins were separately expressed by induction insemi-works after the screening of high expression strains, they were subjected toidentification by SDS-PAGE and mass spectrometric detection.The assemblyelements VP2, VP3and AAP were purified through GE Healthcare Phenyl Sepharose6Fast Flow, GE Healthcare Heparin Sepharose6Fast Flow, GE Healthcare G-25Sepharose Fast Flow and so on.Escherichia coli engineering strains BL21-VP3and BL21-AAP was alsoconstructed. VP3protein was expressed as inclusion body, which was washed anddissolved and then was purified by Ni column. The VP3element expressed byEscherichia coli was used for preparation of polyclonal antibody. AAP was purifiedvia Hydrophobic chromatography column, G-25column and CM column.Conclusion: In contrast to traditional method for rAAV preparation, in this study,rAAV self‐assembly elements, VP2, VP3and AAP, were made up and purifiedrespectively and they are ready for the later in vitro AAV preparation Therefore, tobest of our knowledge, the present study provided a novel approach for large-scale rAAV production without any major problem.