[A novel packaging system of recombinant AAV5/5 vector].
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.
Dong
Xiaoyan
X
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.
Tian
Wenhong
W
Yuan
Zhenhua
Z
Tan
Shuping
S
Wu
Xiaobing
X
D000229:Dependovirus / Q000235:genetics* / Q000502:physiology; D015316:Genetic Therapy; D005822:Genetic Vectors; D057809:HEK293 Cells; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000502:physiology; D006801:Humans; D016865:Reassortant Viruses / Q000235:genetics; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics; D019065:Virus Assembly
D014764:Viral Proteins