Improve

 

New improved part: BBa_K4872013  (pGEX-RV VP7-GII.17-VP1)

Old existing part: BBa_K3992000  (RV VP7)

 

Summary:

 

Figure 1. Expression frame of the plasmid pGEX-RV-GII.17-VP1  constructed

 

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 ), 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.   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] .

 

Figure 2. Plasmid design diagram of pGEX-RV-GII.17-VP1

 

Experimental Approach

In order to construct a recombinant vaccine against norovirus and rotavirus, we first get the RV VP7 and GII.17-VP1 gene, then connect them to the pGEX-4T-1 plasmid by DNA ligase. Finally, we will transform the recombinant plasmid (Figure 2) into E. coli.

 

       

Figure 3. Construction of plasmid pGEX-RV-GII.17-VP1

 

We first amplified the antigen gene RV VP7 and GII.17-VP1 using the PCR amplifier (Figure 3 A). 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 3 B). As shown in Figure 3 C-D, the colony PCR and sequencing results confirmed the successful construction of the plasmid.

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). We examined the expression of the target proteins using SDS-PAGE (Figure 4) and used the ImageJ software to quantify the target band (119 KDa) on the SDS-PAGE gel.

 

Figure 4. SDS-PAGE results of RV-GII.17-VP1 protein under different expression conditions

 

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 a 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.

 

Figure 5 .  SDS-PAGE results of pGEX-RV-GII.17-VP1 protein expression in E. coli Nissle 1917

 

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 (9 k Da) 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.

 

Figure 6. Effect of IPTG induction time and bacterial concentration on protein concentration

 

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:

[1]  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. 

[2]  Desselberger U, Iturriza-Gomara M, Gray JJ. Rotavirus epidemiology and surveillance. Novartis Found Symp 2001: 238:125-C152.

[3]  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.