Part:BBa_K4872011
pGEX-GII.17-VP1
Composite Part: BBa_K4872011 (pGEX-GII.17-VP1)
Construction Design
Plasmid pGEX-GII.17-VP1 is mainly composed of BBa_K4872002 and BBa_K4872003. Correspondingly, for the target gene GII17-VP1, we also used the Tac promoter and Stop codons, and also ligated pGEX-4T-1 as its unloaded load, using zymosynthesis ligation (EcoRI, XhoI) in the process. After that, we transformed the recombinant plasmid pGEX-GII.17-VP1 into E. coli BL21 using heat excitation (Figure 1).
Engineering Principle
GII.17-VP1 gene BBa_K4872002 is the main protein that makes up Norovirus particles.[1] By constructing a recombinant plasmid, GII.17-VP1 can express proteins through IPTG.
Experimental Approach
We first amplified the antigen gene GII.17-VP1 using the PCR amplifier (Figure 2A). Then, the GII.17-VP1 fragment and the pGEX-4T-1 plasmid vector were digested with restriction endonucleases EcoRI and XhoI, followed by the ligation using T4 DNA ligase to obtain the recombinant plasmid pGEX-GII.17-VP1 (Figure 2B). As shown in Figure 2C-D, the colony PCR and sequencing results confirmed the successful construction of the plasmid.
We inoculated the positive transformant and induced protein expression by IPTG. After obtaining protein lysate, we purified the target protein using GST tags and verified the protein expression and purification results by SDS-PAGE. As shown in Figure 3, we successfully expressed and purified GII.17-VP1 protein (60 kDa).
The GII.17-VP1 protein was successfully expressed and purified in this round of experiments, which is expected to be prepared as a norovirus vaccine. In order to develop this norovirus vaccine, the next plan will be focused on the research of the GII.17-VP1 expression in E. coli Nissle 1917 and more evaluation tests shall be made before the implementation. Meantime, we can continue to construct the recombinant plasmid pGEX-RV-GII.17-VP1 to investigate the potential of the bivalent vaccine against norovirus and rotavirus.
References:
- Pogan, R., Weiss, V. U., Bond, K., Dülfer, J., Krisp, C., Lyktey, N., Müller-Guhl, J., Zoratto, S., Allmaier, G., Jarrold, M. F., Muñoz-Fontela, C., Schlüter, H., & Uetrecht, C. (2020). N-terminal VP1 Truncations Favor T = 1 Norovirus-Like Particles. Vaccines, 9(1), 8.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 5497
Illegal BglII site found at 6241
Illegal BglII site found at 6415
Illegal BamHI site found at 6308 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2055
Illegal BsaI site found at 4987
Illegal BsaI.rc site found at 5011
Illegal SapI.rc site found at 342
Illegal SapI.rc site found at 3137
None |