title: Efficient production of an avian adeno-associated virus vector using insect cell/baculovirus expression system.
abstract: Recombinant avian adeno-associated virus (rAAAV) is a promising gene transfer vector for avian cells. Although rAAAV can be produced by co-transfection of HEK293 cells with three plasmids, both scalability and productivity of the transient transfection method can not meet the demand for large-scale in vivo experiments. In this study, a scalable rAAAV production method was established by using insect cell/baculovirus expression system. Three recombinant baculoviruses, namely BacARep, BacAVP and BacAGFP, were generated by transfection of Sf9 cells with the three plasmids expressing AAAV Rep genes, modified VP gene or the inverted terminal repeats-flanked green fluorescent protein (GFP) gene. After demonstration of the correct expression of AAAV genes, rAAAV-GFP was produced by triple infection of insect cells or triple transfection of HEK293 cells for comparison purpose. Electron microscopy revealed the formation of typical AAAV particles in the insect cells. Western blotting showed the correct assembly of rAAAV particles with a VP protein ratio similar to that of AAAV. Quantitative PCR showed that the insect cell-produced rAAAV yield was almost 25-fold higher than that produced by HEK293 cells. Fluorescent microscopy showed that the insect cell-produced rAAAV could transfer GFP reporter gene into two avian cell types with similar transfer efficiency to that of HEK293 cell-produced rAAAV. These data suggest that insect cell/baculovirus expression system could be used for scalable production of rAAAV, and the viral vector produced could be used as the gene transfer vehicle for avian cells.
authors.affiliation: Jiangsu Agri-animal Husbandry Vocational College, Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Taizhou, 225300, China.
authors.forename: Anping
authors.initials: A
authors.lastname: Wang
authors.affiliation: Jiangsu Agri-animal Husbandry Vocational College, Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Taizhou, 225300, China.
authors.forename: Yongjuan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Jiangsu Agri-animal Husbandry Vocational College, Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Taizhou, 225300, China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Wu
authors.affiliation: Jiangsu Agri-animal Husbandry Vocational College, Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Taizhou, 225300, China.
authors.forename: Weiyong
authors.initials: W
authors.lastname: Zuo
authors.affiliation: Jiangsu Agri-animal Husbandry Vocational College, Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Taizhou, 225300, China.
authors.forename: Changming
authors.initials: C
authors.lastname: Guo
authors.affiliation: Jiangsu Agri-animal Husbandry Vocational College, Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Taizhou, 225300, China.
authors.forename: Weiming
authors.initials: W
authors.lastname: Hong
authors.affiliation: Jiangsu Agri-animal Husbandry Vocational College, Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Taizhou, 225300, China. Electronic address: wap4017@163.com.
authors.forename: Shanyuan
authors.initials: S
authors.lastname: Zhu
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D002642:Chick Embryo; D000229:Dependovirus / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D010957:Plasmids / Q000235:genetics; D061987:Sf9 Cells; D018411:Spodoptera; D014162:Transfection / Q000379:methods
chemical_list: D049452:Green Fluorescent Proteins
keywords: Insect cell/baculovirus expression system; Recombinant avian adeno-associated virus