The spike protein of the severe acute respiratory syndrome coronavirus （SARS-CoV） mediates cell fusion by binding to target cell surface receptors. This paper reports a simple method for dissecting the viral protein...The spike protein of the severe acute respiratory syndrome coronavirus （SARS-CoV） mediates cell fusion by binding to target cell surface receptors. This paper reports a simple method for dissecting the viral protein and for searching for foldable fragments in a random but systematic manner. The method involves digestion by DNase I to generate a pool of short DNA segments, followed by an additional step of reassembly of these segments to produce a library of DNA fragments with random ends but controllable lengths. To rapidly screen for discrete folded polypeptide fragments, the reassembled gene fragments were further cloned into a vector as N-terminal fusions to a folding reporter gene which was a variant of green fluorescent protein. Two foldable fragments were identified for the SARS-CoV spike protein, which coincide with various anti-SARS peptides derived from the hepated repeat （HR） region 2 of the spike protein. The method should be applicable to other viral proteins to isolate antigen or vaccine candidates, thus providing an alternative to the full-length proteins （subunits） or linear short peptides.展开更多
基金Supported by the Tsinghua University SARS Special Fund and theNational Key Basic Research and Development (973) Program of China (No. 2003CB716002)
文摘The spike protein of the severe acute respiratory syndrome coronavirus （SARS-CoV） mediates cell fusion by binding to target cell surface receptors. This paper reports a simple method for dissecting the viral protein and for searching for foldable fragments in a random but systematic manner. The method involves digestion by DNase I to generate a pool of short DNA segments, followed by an additional step of reassembly of these segments to produce a library of DNA fragments with random ends but controllable lengths. To rapidly screen for discrete folded polypeptide fragments, the reassembled gene fragments were further cloned into a vector as N-terminal fusions to a folding reporter gene which was a variant of green fluorescent protein. Two foldable fragments were identified for the SARS-CoV spike protein, which coincide with various anti-SARS peptides derived from the hepated repeat （HR） region 2 of the spike protein. The method should be applicable to other viral proteins to isolate antigen or vaccine candidates, thus providing an alternative to the full-length proteins （subunits） or linear short peptides.
