Despite the ever increasing availability of genome sequences from many human pathogens the production of complete proteomes remains a bottleneck. To address this, a high throughput PCR recombination cloning and expression platform has been developed by Feigner and colleagues at the University of California Irvine (UCI). The method relies on high throughput amplification of each predicted open reading frame using gene specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are then expressed in an E. co/r-based cell-free in vitro transcription/translation system and the proteins are printed directly onto microarrays without further purification. The chips can be then used to screen sera from individuals naturally or experimentally exposed to the organisms of interest. The laboratory has completed proteomes for vaccinia virus (203 ORFs) and for F. tularensis (1933 ORFs), and has NIH support to generate the complete B. pseudomallei proteome (-6000 ORFs). This high throughput technology platform for vector generation, protein expression, chip printing and serological analysis will be made available to the RCE. The results of serology from vaccinated or infected, animals and humans are being applied by this laboratory to the problems of vaccine and diagnostic antigen discovery. Both protein subunit vaccines and DNA vaccines are being investigated and developed in this lab. Four potent but non-toxic immunological adjuvants have been acquired, and these adjuvants and vaccine formulations will be available to the RCE through this core. In order to accomplish optimal in vivo expression and immunization with DNA vaccines, it is frequently desirable to prepare mammalian optimized synthetic genes. For this purpose, the resources of the DNA Assembly Core Facility will also be accessible through this core.
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