engleski

Proteome-wide Production of Antibodies against Varicella Zoster Virus

Varicella Zoster virus (VZV) is a highly infectious alphaherpesvirus that causes chickenpox upon primary infection and after reactivation from latency a secondary disease called zoster. The VZV genome is a linear double stranded DNA molecule of approximately 125 kbp, encoding 71 unique proteins. Research on VZV is hampered due to its highly cell-associated nature, restricted host specificity and the lack of an appropriate animal model, as well as the lack of antibody tools. By using the GATEWAY®technology we have cloned all open reading frames (ORFs) of VZV into the pDONR207 vector either in full length or as fragments. The ORFeome in pDONR207 was subcloned into GATEWAY® compatible bacterial or baculovirus expression vectors containing an N- or C-terminal His tag to allow protein purification. Nearly 95% of the VZV proteins could be expressed and purified under denaturing conditions. For several additional proteins, immunogenic peptides were predicted and synthesized. BALB/c mice were immunized with purified proteins or peptides for production of monoclonal antibodies (mAbs). Primary screening of the hybridoma supernatants was done by ELISA and Western blotting on purified proteins followed by Western blotting on lysates of VZV infected cells, and by immunofluorescence. So far, we have successfully produced antibodies against 85% of the VZV proteome. Current efforts focus on mapping the epitopes recognized, isotyping the mAbs and identifying the neutralizing antibodies. This library of VZV specific antibodies is unique, provides a comprehensive tool to understand multiple dimensions of viral and cellular interactions and will be of great importance for both VZV research and diagnostics.

VZV; protein expression; antibody production

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