For plasmid constructions, P. berghei merozoite surface protein 8 (MSP-8, codon-optimized), merozoite surface protein 9 (MSP-9) and influenza matrix protein 1 (M1) genes were cloned, as described previously [15 (link),16 (link),25 (link)]. Codon-optimized P. berghei rhoptry-associated protein 1 (RAP1) gene was acquired from GenScript (Piscataway, NJ, USA).
The recombinant plasmid was transformed into DH10Bac competent cell. Colonies were screened and bacmid DNA from successful clonal construct was extracted using FavorPrep Gel Purification Kit (Favorgen, Cheshire, UK). MSP-8, MSP-9, RAP1, or influenza M1-expressing recombinant baculoviruses (rBVs) were prepared as previously described [26 (link),27 (link)]. Three different VLPs, each expressing one of MSP-8, MSP-9, or RAP1 antigen were generated along with influenza M1 core protein as described previously [26 (link),27 (link)]. After co-culturing Sf9 cells with the rBVs expressing the antigens of interest, culture media were collected at 3 days post-infection (dpi). Cells were pelleted by centrifuging for 1 h, 4 °C, 4800× g. VLPs present within the supernatants were purified and stored at 4 °C as described elsewhere [26 (link),27 (link)].
The recombinant plasmid was transformed into DH10Bac competent cell. Colonies were screened and bacmid DNA from successful clonal construct was extracted using FavorPrep Gel Purification Kit (Favorgen, Cheshire, UK). MSP-8, MSP-9, RAP1, or influenza M1-expressing recombinant baculoviruses (rBVs) were prepared as previously described [26 (link),27 (link)]. Three different VLPs, each expressing one of MSP-8, MSP-9, or RAP1 antigen were generated along with influenza M1 core protein as described previously [26 (link),27 (link)]. After co-culturing Sf9 cells with the rBVs expressing the antigens of interest, culture media were collected at 3 days post-infection (dpi). Cells were pelleted by centrifuging for 1 h, 4 °C, 4800× g. VLPs present within the supernatants were purified and stored at 4 °C as described elsewhere [26 (link),27 (link)].
Free full text: Click here
