Cloning strategies toward the development of a single chain variable fragment (scFv) specific for S. aureus type 5

Abstract

Staphylococcus aureus (S. aureus) is an opportunistic pathogen that is frequently encountered in hospital settings. It is a gram-positive, cluster forming bacteria that causes a variety of infections in humans, including bacteremia. The control of S. aureus infections has become complicated because bacteria have become resistant to a wide range of antibiotics. In these studies we attempted to create a single chain variable fragment (svFv) specific for S. aureus Type 5 capsular polysaccharide (CP) using phage display technology. Hybridomas specific for type 5 S. aureus were grown and total RNA was isolated. cDNA was synthesized and amplified by PCR. Mouse IgM light chain primers (with SacI and HindIII restriction sites) and heavy chain primers (with XhoI and SpeI restriction sites) were used to PCR antibody cDNA and the resulting DNA was purified. Restriction digests of purified TOPO plasmid and PCR products were ligated together, which produced a band about 350-450 base pairs in length. Sequence analysis was performed and the resulting sequence matched a ribosomal protein, not the antibody fragment we were attempting to clone. We believe that the primers that were used to sequence the antibody fragments were too degenerate and thus led to this nonspecific sequence being primed. Further studies will include purifying IgM protein from different hybridomas to determine the amino acid sequence. Using this sequence, more specific primers will be designed to PCR amplify the cDNA from the heavy and light chain of different clones creating a scFv. The scFv will be amplified, cloned into a phagemid vector, and transformed into competent E. coli. Helper phage infection should yield recombinant phage expressing the scFv used to phage g3p (pIII). These recombinant antibodies have the potential of being used to treat S. aureus infections.