Generation of human recombinant antibody Fab fragment and its IgG to adeno-associated virus type II from phage display library / 中华实验和临床病毒学杂志

ABSTRACT

<p><b>BACKGROUND</b>To acquire the recombinant human monoclonal antibodies and IgG to adeno-associated virus type 2 (AAVs-2).</p><p><b>METHODS</b>Construct and pan human Fab antibody library to AAVs-2 was established from normal volunteer donors by using phage display technology and secreted expression in E.Coli system. The positive Fab clones were selected and characterized through ELISA and immunofluorescent assay, and then the heavy and light chain were sequenced. The gene of light chain and heavy chain Fd fragment of recombinant mAb were inserted into baculovirus expression vector pAC-L-Fc and construct expression vectors of intact IgG, then transfected insert sf-9 cell secreted expression in Baculovirus/Insert system. Immunoprecipitation test was used to detect its recognizing region.</p><p><b>RESULTS</b>One clone named AAVs-31 showed positive responses in ELISA and IFA, the Fab was composed of gamma chain and kappa chain IgG was positive in ELISA and IFA. The IgG failed to detect nonassembled or denatured capsid proteins, but recognized the AAVs-2 stock from immunoprecipitation test.</p><p><b>CONCLUSION</b>The authors isolated a clone of Fab and IgG to adeno-associated virus type 2 by phage display technology, they perhaps recognize an epitope which is formed during capsid assembly.</p>