This was initially tested using eGFP as a model antigen however, the wider application of this technology was latterly determined by challenging animals immunised with a novel PsaA-pneumolysin fusion vaccine. PsaA is a 35 kDa

protein detected on the surface of S. pneumoniae that was initially identified as a 37 kDa protein in a non-encapsulated strain. PsaA mTOR inhibitor is a highly conserved protein that is present in over 90 strains tested to date [16]. PsaA has been found to be an effective vaccine candidate in a number of animal models protecting particularly against nasopharyngeal colonisation with concurrent reductions in bacterial counts in bronchial lavage and blood of infected animals [17]. By combining the two antigens, it was hoped to use pneumolysin to effectively deliver PsaA to the mucosal surface and generate protective immunity. GFP from Aequorea victoria was cloned by PCR from pNF320 [18] using the primers 20G and 20H ( Table 1) and inserted into the expression vector pET33bPLY Epigenetics Compound Library research buy [19] to generate pET33bGFPPLY. To create a version of the GFP with enhanced intensity (eGFP), mutations F64L and S65T [20] were created in the original plasmid, pET33bGFPPLY, by site-directed

mutagenesis (Quikchange SDM Kit, Stratagene) using the primers 24W and 24X. This resulted in the production of pET33beGFPPLY. The non-toxic Δ6 version of the plasmid was constructed by site-directed mutagenesis (Quikchange SDM Kit, Stratagene) of pET33beGFPPLY using primers 23B and 23C to introduce the amino acid deletion. To produce a recombinant plasmid expressing eGFP alone, the coding sequence for eGFP was amplified by PCR from pET33beGFPPLY crotamiton using primers 20G and 45L. The resulting product was cut with NheI and SacI, gel purified and ligated into NheI/SacI cut, CIAP-treated pET33b. The resultant plasmid pET33beGFP was transformed into BL21 cells. PsaAPly fusion constructs were generated using In-fusion technology cloning (Clontech, France). In brief, PsaA gene was amplified from genomic DNA

from S. pneumoniae TIGR4 using primers 65Y and 66A. Similarly, PLY was amplified form pET33bPLY using primer 65W and 65X. To allow In-fusion cloning to proceed purified pET33b(+) plasmid was digested with BamHI and HindIII restriction enzymes at 37 °C for 3 h. The cut plasmid and all the PCR products were cleaned using gel purification kit (Qiagen) and DNA quantity and quality was measured by Nanodrop 1000 spectrophotometer (Thermo Scientific, UK). Once relative quantities of DNA had been established, 100–150 ng of restriction enzyme-digested, gel-purified pET33b(+) and each DNA PCR amplified fragment were mixed at a molar ratio of 1:2 in a total volume of 10 μL in one tube of In-Fusion Dry-Down reaction mix (Clontech, France). The reaction was incubated 15 min at 37 °C, followed by 15 min at 50 °C. The samples were then transferred to ice, and diluted 1:5 by the addition of Tris EDTA (TE) buffer.