| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1995: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1994: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1993: ¥700,000 (Direct Cost: ¥700,000)
|
|---|
| Research Abstract |
For establishment of in vitro packaging system, recombinant-adeno-associated virus (r-AAV) producing HeLa cell lines were established by cotransfection of packaging plasmid (psub201) and vector plasmid, (pNAV/AAV ; neoR driven by HSV-TK promoter). After G418 selection, neoR colonies were screened by PCR with AAV-rep primer, and analyzed for the abilities to produce rAAV by Adenovirus (Ad) infection, and genomic Southern blots with probes of AAV-cap and neoR.Packaging and vector plasmids were integrated into the host genomes and the titers of the rescued rAAV increased in parallel with copy numbers of both plasmids. The expression of rep and cap gene was confirmed in an Ad dependent manner by Northern analysis. The titer of rAAV in the cell-lysate from the highest titer cell line (HAN10) was 10^<5-6> cfu/10^6 cells.
Packaging HeLa cell lines were also generated by cotransfection of packaging plasmid and plasmid containing selectable gene (hygR). These cell lines can be utilized to genera
… More
te the favorite rAAV producing cell line by transfection of various vector plasmid, or transduction of various rAAV.In the case of pNAV/AAV or neoR-rAAV,the titers of rAAV in the cell-lysate from the cloned cell lines were 10^<5-6> and 10^4 cfu/10^6 cells, respectively. The lysate of these r-AAV producing and packaging cell lines were useful for invitro r-AAV packaging system as the reaction medium.
The truncated AAV-cap gene (0.7kb ; 23kDa) was inserted downstream from the maIE gene of E.coli, which encodes maltose-binding protein (34kDa ; MBP) in E.coli. expression vector pMAL-c. This plasmid contains the sequence coding for the recognition site of the specific proteinase factor Xa downstream of the polylinker sites. After trans-formation to JM109, the truncated AAV-Cap protein were expressed by 10mM IPTG induction as the fusion protein of MBP.But the purified AAV-Cap protein could not be separate by the digestion of factor Xa, and the fusion protein of AAV-Cap protein were used to prepare the polyclonal and monoclonal antibodies.
r-AAV in the cell-lysate was quantitatively adsorbed to the sulfated cellulose affinity chromatography, and r-AAV was quantitatively recovered with 0.1MNaCl in phosphate buffer pH7.2. Using the affinity chromatographic technic, neoR-rAAV from HAN10 was partially purified, changed the solvent from cell culture medium to phosphate buffer, and concentrated to 10^8cfu/ml. This affinity chromatographic technic is convenient for the isolation and concentration of r-AAV from the in-vitro packaging system.
The r-AAV producing and packaging cell-lines, the polyclonal and monoclonal antibodies against AAV-Cap protein, and the sulfated cellulose affinity chromatography technic may be essential for establishment of the in-vitro r-AAV packaging system. Less
|
