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A novel packaging system of recombinant AAV5/5 vector / 生物工程学报
Chinese Journal of Biotechnology ; (12): 679-686, 2010.
Article Dans Chinois | WPRIM | ID: wpr-292220
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.
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Texte intégral: Disponible Indice: WPRIM (Pacifique occidental) Sujet Principal: Physiologie / Recombinaison génétique / Protéines virales / Thérapie génétique / Virus recombinants / Herpèsvirus humain de type 1 / Dependovirus / Assemblage viral / Cellules HEK293 / Vecteurs génétiques Limites du sujet: Humains langue: Chinois Texte intégral: Chinese Journal of Biotechnology Année: 2010 Type: Article

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Texte intégral: Disponible Indice: WPRIM (Pacifique occidental) Sujet Principal: Physiologie / Recombinaison génétique / Protéines virales / Thérapie génétique / Virus recombinants / Herpèsvirus humain de type 1 / Dependovirus / Assemblage viral / Cellules HEK293 / Vecteurs génétiques Limites du sujet: Humains langue: Chinois Texte intégral: Chinese Journal of Biotechnology Année: 2010 Type: Article