Clearing, amplification and activity detection of the recombinant adeno-associated virus vector2/1with adiponectin

VernacularTitle:rAAV2/1-Acrp30病毒的纯化、扩增与活性检测
Author: Qiangxiang LI ; Huiju ZHONG ; Yangshi OU ; Huaqing TAN ; Min WANG ; Guoxiang LONG ; Guo LI
Publication Type:Journal Article
Keywords: rAAV2/1-Acrp30; Amplification; Activity detection
From: Chinese Journal of Microbiology and Immunology 2009;29(7):618-622
CountryChina
Language:Chinese
Abstract: Objective To clear, amplify and detect the activity of the recombinant adeno-assoeiated virus vector with adiponectin( rAAV2/1-Aerp30 ). Methods Recombinant plasmid pSNAV2.0-Acrp30 was obtained. The recombinant plasmid was then transfected into BHK21 cells using LipofectAMINETM 2000. The G418 resistant cells were obtained consequently. These cells were infected with HSVI-rc/△UL2 which has the function of packaging and copying recombinant AAV. After purification, the construction of recombinant rAAV2/1-Aerp30 was collected. Results The construction of recombinant pSNAV2.0-Acrp30 was confirmed by PCR electrophoresis and digestion with restriction enzyme. The gene sequencing showed that the recombinant pSNAV2.0-Acrp30 was correct. The virus titer was about 1.0×1012 μg/ml. The purity f the recombinant AAV2/1 was fairly high using the SDS-PAGE method. Conclusion With this method, rAAV2/-Aerp30 with high virus titers and purity can be acquired successfully and it can meet the demands of the experimental study of Acrp30 gene therapy of GK rats.