An Optimized Low-Endotoxin Plasmid Extraction Method for AAV Vector Packaging
HE Jinsheng, LU Yizhen, ZHANG Yuxue, WEI Yalu, GAO Haoshen, HE Rongyan, ZHANGSUN Dongting, LUO Sulan*
This study aimed to establish a low-cost, high-efficiency, transfection-grade plasmid extraction method with low endotoxin levels, suitable for AAV (adeno-associated virus) vector packaging. Based on the principle of phase separation via cloud point extraction, the traditional alkaline lysis buffer system was optimized by integrating lysis and endotoxin removal into a simplified workflow. The quality, endotoxin level, and biological activity of the extracted plasmids were comprehensively evaluated using UV spectrophotometry, restriction enzyme digestion, agarose gel electrophoresis, limulus amebocyte lysate assay, and quantitative real-time PCR. The performance of the method was also compared with that of commercial plasmid purification kits. The results demonstrated that this method yields plasmids with high productivity, good purity, and intact sequences, with endotoxin residues below 1 EU/μg, meeting the requirements for AAV packaging. The resulting AAV vectors exhibited good in vitro activity. Notably, the cost of endotoxin removal per extraction representing a reduction of over 85% compared to commercial reagents, and the protocol shortened the extraction time by approximately 30 min. This method significantly simplifies the operation while ensuring plasmid quality and offers practical value and broad applicability for AAV vector production.
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