Part:BBa_K3992000

VP7

Improvement by 2023 iGEM Team Canton-HS

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), 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].

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

Experimental Approach

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 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.

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 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.

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 (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).

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

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:

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: 238:125–152.
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.

VP7

Usage and Biology

Otavirus (RV) is the main viral pathogen that causes severe acute diarrhea in infants and young children. Almost all children under five weeks of age have been infected with the virus, causing nearly 130,000 deaths worldwide each year. Social conditions in developing countries have led to reduced effectiveness of oral rehydration solutions and vaccines, as well as a lack of approved antiviral drugs, making rotavirus infection a global health problem. RV structural protein vp7, on the outermost layer of virus particles, is the first choice for the development of genetic engineering vaccines. We are trying to develop a new oral vaccine for hand, foot and mouth disease due to its advertisement for infants and young children.

Polymerase Chain Reaction

A PCR verification was performed to confirm whether the sequence of synthesized VP7-LTB is cerrect. Agarose gel electrophoresis was used to assess the PCR’s result. According to the 15000 bp DNA marker, the PCR amplified DNA fragments possess the desired right size.

Figure 1. PCR verification of VP7 and VP7-LTB.

Proof of function

SDS PAGE

Figure 2 shows the protein expression of E. coli with SDS-PAGE. Our purpose was to identify the presence of new proteins and confirm whether they are our proteins of interest --- VP7.

Figure 2 SDS-PAGE of BL21 protein expression.

There are supernatants and precipitates. We suggest that the virus-induced proteins existed in the form of insoluble inclusion body. Compared the right figure with the other one, we could find that the adding of LTB stimulates the expression of VP7

Figure 3 SDS-PAGE of WB800n protein expression.

VP7 and VP7-LTB were successfully expression in E. coli predominantly as inclusion bodies. The protein expression in B. subtilis was not successful as indicated by SDS-PAGE and Western blot These graphs show the relationship between time and protein expression

Figure 4 VP7 expression with 1mM IPTG.

Figure 5 VP7 expression with 2 mM IPTG.

Conclusion: VP7 was present in both the supernatant and sediment, but predominantly as inclusion bodies in the sediment.

References

7.Offit Paul A. Challenges to Developing a Rotavirus Vaccine.[J]. Viral immunology,2018,31(2).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1
  Illegal BamHI site found at 799
  Illegal XhoI site found at 893
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1
  
• 1000
  COMPATIBLE WITH RFC[1000]