We are the Bis-Vacc team. Our team is committed to developing a novel EV71/RV combined vaccine. We searched the iGEM Biological Parts library for BBa_K4004001, IL12. This is a basic part built by iGEM11_Brazil-France in 2011 to vaccine adjuvant. Therefore, this year, our team not only constructed a new composite part BBa_K4276007, but also added new experimental data for the part BBa_K554005, IL12 . It added the data part to provide data support for future teams.
Moreover, we propose the ideas that VR-vp7 and EV71-vp1 are expressed in tandem by fusing with the membrane binding protein GLBP to display on the surface of bacteria and adding the vaccine adjuvant IL-12 to make the vaccine stable expression at the same time. Finally, the green fluorescent protein (GFP) tag was added to facilitate the detection of the localization of vp7-vp1 in bacteria. It can provide new viewpoints on vaccines about EV71 and RV for future iGEM teams.
For mucosal immunity, intranasal IL-12 stimulates mucosal immunity along with antigen-specific immune responses and stimulates the production of small amounts of IFN-γ, which induces the production of specific serum IgG and mucosal IgA antibodies and is safer and more effective than conventional LTB adjuvants.
Figure 1.The PCR identification result of IL12 The bright band of the picture is 1500bp, which proves to be IL12. The results show that we have successfully obtained the IL12 fragment(Figure 1).
vp1 and vp7 are fragments screened from EV71 and RV, which need chimeric recombination to label GFP. And the plasmid added the membrane binding protein GLBP to bind the protein to the bacterial surface.
Figure 2. The map of the plasmid pET28a-GLBP-vp7-vp1-IL12-GFP.
We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 3), and the GLBP-vp7-vp1-IL12-GFP was successfully constructed.
Figure 3. The sequencing blast results of the GLBP-vp7-vp1-IL12-GFP.
In order to obtain the GLBP-vp7-vp1-IL12-GFP protein, we transferred the plasmids into E.coli BL21(DE3). Figure 4 demonstrated that the GLBP-vp7-vp1-IL12-GFP was successfully transformed into E.coli BL21 (DE3).
Figure 4: The PCR identification result of GLBP-vp7-vp1-IL12-GFP. We expanded the culture in the LB medium, and added IPTG to induce protein expression when the OD600 reached 0.4. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used nickel column to purify the GLBP-vp7-vp1-IL12-GFP protein we wanted.
Figure 5. The SDS-PAGE result of the four proteins In Figure 5, we found the GLBP-vp7-vp1-IL12-GFP protein in lane S, P and E, it indicated that the GLBP-vp7-vp1-IL12-GFP protein successfully expressed in E.coli BL21 (DE3).
This new composite part is a technical innovation of the project as a new attempt.
