A novel packaging system of recombinant AAV5/5 vector.
- Author:
Xiaoyan DONG
1
;
Wenhong TIAN
;
Zhenhua YUAN
;
Shuping TAN
;
Xiaobing WU
Author Information
1. State Key Laboratory for Molecular Virology and Genetic Engineering, Institute for Viral Disease Prevention and Control, Chinese Center for Disease Prevention and Control, Beijing 100052, China.
- Publication Type:Journal Article
- MeSH:
Dependovirus;
genetics;
physiology;
Genetic Therapy;
Genetic Vectors;
HEK293 Cells;
Herpesvirus 1, Human;
genetics;
physiology;
Humans;
Reassortant Viruses;
genetics;
Recombination, Genetic;
Viral Proteins;
genetics;
Virus Assembly
- From:
Chinese Journal of Biotechnology
2010;26(5):679-686
- CountryChina
- Language:Chinese
-
Abstract:
We developed a scalable AAV5/5 vector packaging system by using replication competent recombinant herpes simplex type 1 virus as helper virus. The fragment containing rep and cap genes of AAV5 was inserted into the non-necessary gene (UL2) of HSV1 genome, resulting in the helper virus rHSV1-rep5cap5. An AAV5/5 vector pAAV5neo carrying two AAV5 ITRs was constructed by inserting a neo gene expression cassette into the plasmid backbone of pAV5CMV-GFP. pAAV5neo-EGFP was constructed by inserting EGFP gene into pAAV5neo. BHK21 cell was transfected with pAAV5neo-EGFP and cultured in the presence of G418. EGFP expression positive monoclonal cells were picked up, and one that produced rAAV5/5-EGFP with the highest efficiency under the help of rHSV1-rep5cap5 was chosen as the production cell line named as C020. rAAV5/5-EGFP was produced by infecting C020 cells with rHSV1-rep5cap5, and crudely purified by our previous method of 'chloroform treatment-PEG8000/NaCl precipitation- chloroform extract'. rAAV5/5-EGFP preparation with high purity was obtained by ultrafiltration with molecular weight cut-off value of 100 kDa. SDS-PAGE stained with Coomassie brilliant blue R250 showed clearly specific pattern of three bands of AAV capsid proteins. rAAV5/5-EGFP was also assayed using negative stain transmission electron microscopy and the majority of the virus particles were found solid. About 30% green fluorescent cells could be seen after infecting HEK293 cells with rAAV5/5-EGFP 24 h at 1 x 10(5) vg/cell. In conclusion, we have established an efficient AAV5/5 vector production system and could produce recombinant AAV5/5 virus in large amounts for gene therapy research.
