P117: Using splicing overlap extension to create a FliC:GP132 recombinant protein

Genetically similar to Ebola virus, Marburgvirus (MARV) causes hemorrhagic fever with high lethality. This health threat has no cure; consequently, research is aimed towards vaccine development. One method employed to amplify immune response is the creation of synthetic proteins that combine established bacterial antigens with viral epitotes. An epitote for MARV called GP132 for the 15 amino acids starting at amino acid 132 in MARV glycoprotein has been found to produce a cellular immune response. Highly conserved regions in flagellin serve as pathogen-associated molecular patterns (PAMPs) for toll like receptor 5 (TLR5) recognition. Each flagellin monomer is composed of four domains: D0, D1, D2, and D3. The D1 region is essential for TLR5 recognition. A fusion protein from the Salmonella typhimurium flagellin monomer FliC and GP132 was created using splicing overlap extension (SOE). This method provides an alternative to sequence dependent restriction digestion. SOE utilizes polymerase chain reaction (PCR) to create the desired DNA sequence without the sequence dependent limitations inherent to restriction enzyme digest. Primers are designed with complementary overlap regions. When products from separate PCR runs using these primers are combined, the overlapping regions anneal creating a new recombinant template. Sequential PCR runs join multiple fragments until the desired recombinant sequence is achieved. This method was used to insert GP132 within the D3 domain of FliC. The resulting protein will be tested for TLR5 activity.