Part:BBa_K4872001

GII.4-VP1

Contribution by 2023 iGEM Team Canton-HS

Basic Part: BBa_K4872001 (GII.4-VP1)

Profile

• Name: GII.4-VP1
• Base Pairs: 1623 bp
• Origin: Norovirus GII, Synthetic
• Properties: Norovirus is an envelope-free icosahedron composed of single-stranded positive-stranded RNA, with VP1 forming the viral capsid. VP1 is the main protein that makes up virus particles.

Usage and Biology

The main capsid (VP1) protein of NoV is believed to be closely related to the infectivity and antigenicity of these strains[1]. Many previous reports have shown that the NoV VP1 gene has rapidly evolved, resulting in significant differences in antigenicity[2-4]. Early findings also indicate that the rapid evolution of the VP1 gene in NoV is closely related to the gastroenteritis pandemic caused by NoV [5].

Cultivation, Purification, and SDS-PAGE

Figure 1. Plasmid design diagram of pGEX-GII.4-VP1

We used the Tac promoter (BBa_K4872004), which is suitable for E. coli protein production, for transcription of the expression frame. At the C-terminus of target gene GII.4-VP1, we fused a GST tag (BBa_K608408), which facilitates soluble expression of the protein and can be used to purify the protein (Figure 1). After obtaining this recombinant plasmid, we transformed it into E. coli BL21(DE3). Finally, we used IPTG to induce the expression of the target protein GII.4-VP1.

Figure 2. Construction of plasmid pGEX-GII.4-VP1

Firstly, we amplified the antigen gene GII.4-VP1 using the PCR amplifier (Figure 2A). Then, the GII.4-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.4-VP1 (Figure 2B). As shown in Figure 2C-D, the colony PCR and sequencing results confirmed the successful construction of the plasmid.

Figure 3. SDS-PAGE results of pGEX-GII.4-VP1 protein expression

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. The results are shown in Figure 3, we successfully expressed GII.4-VP1 protein (60 KDa), but the purified protein was barely visible, which might be due to the low expression of protein.

Since the low expression of the protein was not successfully purified, we need to explore the optimal expression conditions for the purification of this protein to increase the expression level of the protein. After we solve this problem, we could further step to continue the construction of recombinant plasmid pGEX-RV VP7-GII.4-VP1.

Reference:

1. Debbink, K., Lindesmith, L. C., Donaldson, E. F., & Baric, R. S. (2012). Norovirus immunity and the great escape. PLoS pathogens, 8(10), e1002921.
2. Yang, Y., Xia, M., Tan, M., Huang, P., Zhong, W., Pang, X. L., Lee, B. E., Meller, J., Wang, T., & Jiang, X. (2010). Genetic and phenotypic characterization of GII-4 noroviruses that circulated during 1987 to 2008. Journal of virology, 84(18), 9595–9607.
3. Bok, K., Abente, E. J., Realpe-Quintero, M., Mitra, T., Sosnovtsev, S. V., Kapikian, A. Z., & Green, K. Y. (2009). Evolutionary dynamics of GII.4 noroviruses over a 34-year period. Journal of virology, 83(22), 11890–11901.
4. Zakikhany, K., Allen, D. J., Brown, D., & Iturriza-Gómara, M. (2012). Molecular evolution of GII-4 Norovirus strains. PloS one, 7(7), e41625.
5. Robilotti, E., Deresinski, S., & Pinsky, B. A. (2015). Norovirus. Clinical microbiology reviews, 28(1), 134–164.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 612