Part:BBa_K4872013
pGEX-RV VP7-GII.17-VP1
Summary:
We have made improvements on the original components BBa_K3992000. Throughout the design, we used tac promoter (BBa_K4872004) to induce protein expression and added NoV GII.17-VP1 (BBa_K4872002), which is the main protein that makes up Norovirus particles[1]. We connected these two fragments GII.17-VP1 and RV VP7 to the same vector pGEX-4T-1 (BBa_K4872003) through GS-linker to express a fusion protein.
Construction Design and Engineering Principle
Rotavirus belongs to the family Reoviridae. The virion is a triple-layered particle consisting of six structural proteins encoded by 11 segments of double-stranded RNA that can be separated on a polyacrylamide gel.[11] The outer layer consists of two neutralizing antigens, namely a protease-sensitive P-type antigen (VP4) and glycoprotein G type antigen (VP7). The antigenic and molecular properties of these surface proteins have been used to classify rotavirus strains[2]. VP7 is a glycoprotein and determines the G serotype[3].
Experimental Approach
We first amplified the antigen gene RV VP7 and GII.17-VP1 using the PCR amplifier (Figure 3A). Then, the RV VP7 and GII.17-VP1 fragments underwent homologous recombination with the pGEX-4T-1 plasmid vector to obtain the recombinant plasmid pGEX-GII.4-VP1 (Figure 3B). As shown in Figure 3C-D, the colony PCR and sequencing results confirmed the successful construction of the plasmid.
Finally, we transformed plasmid pGEX-RV-GII.17-VP1 into E. coli Nissle 1917. Through the SDS-PAGE gel map, it was found that we have weak bands within the 90 kDa range, which means that RV-GII.17-VP1 protein (119 KDa) expression level is weak in E. coli Nissle 1917 (Figure 5). This initially confirmed that RV-GII.17-VP1 could be successfully expressed in Nissle 1917, but the expression conditions need to be optimized.
The expression of RV-GII.17-VP1 in E. coli Nissle 1917 was low, so we need to improve and optimize the protein expression method to achieve large-scale expression of RV-GII.17-VP1. But we did see the potential of this bivalent vaccine against norovirus and rotavirus, no doubt this research will be continually developed and is a promising product in the vaccine market for society.
Characterization/Measurement
We inoculated the transformant containing pGEX-RV-GII.17-VP1, induced its expression, and explored its optimal expression conditions. To find the optimal conditions for the highest protein expression, we chose different concentrations (OD600=0.3/0.6/0.8/1) of the bacterial solution and different IPTG induction times (0 h/4 h/8 h/16 h). After obtaining each protein lysate, we measured the total protein amount (A280) under different induction conditions (Figure 6A). In addition, we examined the expression of the target proteins (119 kDa) using SDS-PAGE (Figure 4) and used the ImageJ software to quantify the target bands on the SDS-PAGE gel, collected and organized the data, and plotted a line graph with OD600 as the x-axis and gray value as the y-axis (Figure 6B).
As shown in Figure 6, the protein concentration roughly tended to increase with increasing bacterial concentration at the same IPTG induction time. When the bacterial concentration was 0.8 and the induction time was 8 hours, the best protein expression level was achieved, indicating that this condition was more suitable for expressing more target proteins.
Compared to part BBa_K3992000, we constructed a fusion protein RV VP7-GII.17-VP1. In terms of performance, it can not only target rotavirus but also norovirus, which can be used to develop a combination vaccine of rotavirus and norovirus. When the bacterial concentration is 0.8 and the induction time is 8 hours, the protein expression level is the best, indicating that this condition is more suitable for expressing more proteins. This condition can be better applied to subsequent vaccine development.
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.
- Desselberger U, Iturriza-Gomara M, Gray JJ. Rotavirus epidemiology and surveillance. Novartis Found Symp 2001: 238:125–152.
- Gentsch JR, Laird AR, Bielfelt B, et al. Serotype diversity and reassortment between human and animal rotavirus strains: implications for rotavirus vaccine programs. J Infect Dis. 2005;192 Suppl 1:S146-S159.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 6386
Illegal BglII site found at 7130
Illegal BglII site found at 7304
Illegal BamHI site found at 5690
Illegal BamHI site found at 7197 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2055
Illegal BsaI site found at 5876
Illegal BsaI.rc site found at 5900
Illegal SapI.rc site found at 342
Illegal SapI.rc site found at 3137
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