Proof of Concept
1. We established stable Leishmania transfectants and
proved the function of promotor with hygromycin drug selection gene
The promoter and the ribosomal binding site of Leishmania genome has not been elucidated yet. We selected the 5'- untranslated region of a highly expressed gene, P36, to be the promoter, RBS binding site and other extra function needed for Leishmania protein expression. We also added a hygromycin resist gene to serve as a dual functional biobrick of regulatory and selection marker. We provide the user with the regulation of protein expression and also the drug selection system that is most commonly and effectively used in Leishmania experiments. The pSB1C3-5'HYG-OVA-3'UTR was transfected into Leishmania 12-DT strain, and the transfectants were selected by hygromycin. We obtained the hygromycin-resistant Leishmania transfectants (Figure 1. B, C). In the negative control group (Figure 1. A), we could see that almost all cells were dead and the shape of Leishmania was not normal. It showed that Leishmania transfectants were resistant to high concentration of hygromycin. The function of 5’HYG was assayed more precisely in figure 2.
2. Successfully expressed Hemagglutinin of H1N1
Since we are performing immunology experiments, it is very important that the antigen is correctly expressed. We used BL21 competent cell to express the HA sequence and detect the protein by Western blot analysis.
The pSB1C3-J04500-HA can is checked by Western blot analysis. We successfully recognize the HA protein from Western blot analysis (approximately 62.3kd) when induced by IPTG (Figure 3.).
3. The antibody production of Leijuvant in mice
The IgG1 antibody titer increased drastically after the second boost on the 15th day. And the antibody titer reached to peak on the 25th day (Figure 4A.). Inactivated Leishmania can achieve over 60% of the antibody titer. As for OVA-expressing inactivated Leishmania, the antibody titer can reach to 70% on the 25th day. The IgG2a antibody titer of inactivated Leishmania increased extremely on the 20th day and is significantly higher compare to Alum on the 25th day(Figure 4B.). Leijuvant is a potential bi-pathway adjuvant that can stimulate both Th1 and Th2 immune responses.