engleski

HIGH-THROUGHPUT EXPRESSION OF THE VARICELLA-ZOSTER VIRUS (VZV) PROTEOME FOR SERUM PROFILING EXPERIMENTS AND THE PRODUCTION OF MONOCLONAL ANTIBODIES

Varicella-Zoster virus (VZV) is a member of the neurotropic alphaherpesvirus subfamily. VZV causes chickenpox upon primary infection and may cause shingles after reactivation from latency. The VZV genome consists of approximately 125kbp and encodes at least 71 unique proteins. We have cloned all 71 known viral ORFs by recombinatorial cloning (RC) into the GATEWAY&reg ; entry vector pDONR207. Viral ORFs encoding membrane proteins have been cloned both as full length clones, and as domains lacking the respective transmembrane domains. All ORFs and ORF domains were cloned without a stop codon, enabling the construction of C-terminal fusion proteins. Altogether 96 entry vectors were constructed and verified by sequencing. In parallel a portfolio of N- and C-terminally tagged, GATEWAY&reg ; compatible bacterial expression vectors have been constructed. These customized, pET-based destination vectors enable the efficient subcloning of the respective viral ORFs and ORF domains and their bacterial expression and purification as fusion proteins. Utilizing N-terminally RGS-His and N-terminally MBP tagged bacterial expression vectors we were able to express and purify some 75% of the VZV proteome in small scale. In initial serum profiling experiments, the purified recombinant proteins were dot blotted on nylon membranes and probed with serum samples derived from VZV-positive and – negative persons. The result of this pilot experiment indicated that the VZV proteins encoded by ORF14 (gC), ORF22 (large tegument protein), and ORF68 (gE) are predominantly targeted by the human humoral immune response. Some 40% of all VZV proteins have been purified in large scale (>500&micro ; g) utilizing the above mentioned tags and used to immunize mice for monoclonal antibody (mAB) production purposes (ongoing). We could develop mAB's against 20% of all VZV proteins(ongoing). The resulting mAB's have been tested in WB and IF and will provide a valuable tool for studying the respective viral proteins in detail.

VZV; mAbs; serum profiling

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