| As a gene transfer vector, Adeno-associated virus (AAV) has many advantages, including stable expression of foreign genes, infection of a multiple cell types, nonpathogenicity to the hosts, low immunogenicity. Recombinant AAV has gotten more and more attention in gene therapy in recently years. Since Myers and Carter proposed AAV assembly pathway in1980, the research on the viral genome replication has made great advance, whereas molecular events underlying the capsid formation and genome encapsidation are largely not clear. AAV genome contains two larger open reading frames (ORFs), encoding the Rep and Cap proteins, respectively. The cap ORF1encodes the structural proteins VP1, VP2and VP3, while the ORF2encodes adeno-associated virus assembly-activating protein (AAP), which is essential for the capsid assembly process.In this study, the coding sequences for the AAP of AAV type5(AAP5) and the VP3of avian adeno-associated virus (AVP3) were amplified by PCR, and cloned individually into prokaryotic expression vector pET-30a. The recombinant plasmids were transformed into competent BLR E.coli.and expression of the proteins were induced with IPTGThe approximate molecular weight of AAP5protein was44KD, most of which was soluble. The approximate molecular weight of AVP3protein was81KD, most of which was expressed as the insoluble inclusion bodies.By using Ni-NTA affinity column, both AAP5and AVP3proteins were purfied from the recombinant bacterial lysate and used to immunize BalB/C mice. After3times immunization, the ELISA antibody titers reached tol:102400and1:51200, respectively. The spleen cells were prepared and fused with SP2/0myeloma cells. Then the positive cell clones were screened by ELISA detectionusing the pured proteins as the antigens. After3cycles of subcloning, four hybrydoma cell clones secreting the specific antibody aganst AAP5or AVP3were established. After injection into mice, the antibody titer to AAP5was216and to AVP3was29. |
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