There is a summary of the background of vp1 and vp7 protein-related parts. For the production of the combined vaccine, we searched the iGEM Biological Parts library for similar fragments of EV71 and RV, and selected BBa_K4004001, VP-1. This is a basic part built by the iGEM21_Shanghai_Metropolis team in 2021, they tried to fuse VP-1 with vaccine adjuvant LTB, protein expression.
To optimize the oral-vaccine project, we fused vp1 and vp7 and inserted a membrane-bound protein BBa_K2328010, GLBP to allow the fusion protein to bind to the bacterial surface. To facilitate the detection of vp7-vp1 localization in bacteria, a green fluorescent protein (GFP) was added. Finally, we have constructed the plasmid pET28a-GLBP-vp7-vp1-GFP to protect children from two viruses EV71 and RV.
Moreover, the exogenous protein GLBP-vp7-vp1-GFP does not need to be purified and can be taken directly with the bacteria. It is safe and free of endotoxin and will be a promising infant vaccine.
The researches indicate that the major capsid protein vp1 is the main component of the polyomavirus capsid and the main antigens of EV71. vp1 monomers are typically about 350 amino acids in length and can self-assemble into icosahedral structures consisting of 72 pentamers of 360 vp1 molecules. A capsid protein, vp7, is the main antigens of RV, early studies using sera from hyperimmunized animals in cross-neutralization assays described some different serotypes that were found to infect humans and animals based on the vp7 protein. The virus is assigned a G-type, referring to the glycosylated structure of the protein. GLBP is a membrane protein belonging to the family of ATP-binding cassette transport proteins. It is widely expressed in the cell wall of wild-type Bifidobacteria and actively transports specific substances across the cell membrane of all organisms using the energy of adenosine triphosphate (ATP). Antigens expressed in fusion with GLBP can induce mucosal immunity and immune memory. Green fluorescent protein (GFP) is a fluorescent tag that facilitates the detection of vp7-vp1 localization on Bifidobacteria.
Our goal is to produce vp1 and vp7 combined vaccine to protect infants from these two viruses. In addition, the oral method is much more convenient, which can not only improve the vaccination rate, but also does not require professionals to vaccinate, and the consumption of social resources is lower.
First, we amplify VP7, VP1, GLBP, and GFP by PCR. The results represent that the amplification of VP7, VP1, GLBP, and GFP is successful (Figure 1(A, B)). Then, we overlap PCR to amplify GLBP-VP7, and VP1-GFP by PCR. The PCR results show that the fusion of GLBP- VP7 and VP1-GFP is successful (Figure 1(C)). Finally, the GLBP-vp7 and vp1-GFP were fused by overlap PCR and the PCR results indicate that the fusion of GLBP-vp7-vp1-GFP is successful (Figure 1(D)).
Figure 1. The PCR identification result of DNA fragments. The fragments GLBP-VP7-VP1-GFP were double-enzyme digesed, and inserted into the XbaI and BamHI site of pET28a (+) vector, to obtain the recombinant plasmids pET28a-GLBP-VP7-VP1-GFP. Then the pET28a-GLBP-VP7-VP1-GFP transform into E.coli DH10B, the colony PCR identification results show that transformants 2, 4, 7, and 9 have right size (Figure 2).
Figure 2. The PCR identification result of pET28a-GLBP-vp7-vp1-GFP in E.Coli DH10B. Thus, we picked up positive colonies to sequence in Genwiz. The result showed as Figure 3 and the sequence well matched with the template.
Figure 3. Sanger sequencing of pET28a-GLBP-vp7-vp1-GFP plasmid
We have extracted the recombinant plasmids pET28a-GLBP-VP7-VP1-GFP from DH10B, and transferred them into BL21 (DE3) , added IPTG to induce protein expression when the OD600 reached 0.3-0.5. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins pET28a-GLBP-VP7-VP1-GFP. Next, we used nickel column purification to purify the protein pET28a-GLBP-VP7-VP1-GFP we wanted.
Figure 4. SDS-PAGE gel demonstrated the expression of the protein samples. The negative control is pET28a plasmid without exogenous gene. The expression of GLBP-vp7-vp1-GFP was confirmed by SDS-PAGE, as shown in Figure 4.
This image showed the GLBP-VP7-VP1-GFP expressed in the bacteria.
Figure 5. The comparison of supernatant of GLBP-VP7-VP1-GFP and without GFP
The left tube is bacteria expressing pET28a-VP7-VP1 without GFP as the negative control. The right tube is bacteria expressing pET28a-GLBP-VP7-VP1-GFP. The lift panel showed the supernatant is under visible light, and the right panel showed the supernatant is under blue light.
Figure 6. Fluorescence microscopy of GLBP-VP7-VP1-GFP.
It’s obvious that the supernatant contains lots of protein tagged GFP after ultrasonic crushing bacteria expressing pET28a-GLBP-VP7-VP1-GFP, which indicating the VP7 and VP1 overexpressed in the bacteria and bond to the cell surface.
At the same time, we monitored the bacteria expressed the VP1 and VP7 for 0 to 8 hours using GFP intensity. as shown in figure 7, the result exhibited the relationship of protein expression with time and the effect of IPTG on fluorescence intensity. The longer induction time is beneficial for protein expression, but according to the result, 3 hours is enough for VP1 and VP7 expression. In addition, the IPTG concentration has a significant influence on the GFP intensity during the cultivation. Concentration of IPTG in 0.25 to 10 mM has a little effect on the protein expression. However, higher concentration of IPTG (10 mM) increased the GFP intensity.
Figure 7. The three-dimensional model diagram of the time-IPTG concentration-GFP surface.
Compared to the 2021 oral vaccine project, we designed the plasmid pET28a-GLBP-vp7-vp1-GFP which fused VP1 and VP7 and added a membrane-bound protein (GLBP) to allow the fusion protein to bind to the bacterial surface. To facilitate the detection of vp7-vp1 localization in bacteria, a green fluorescent protein (GFP) was added. Moreover, we successfully detected the fluorescence signal of GLBP-vp7-vp1-GFP. And the model was fitted with the data of IPTG-induced protein GLBP-vp7-vp1-GFP. The results showed that the main antigens of EV71 and RV: vp1 and vp7 were co-expressed, and fused with GLBP. and expressed on the surface of bacteria to obtain a combined bivalent vaccine. This innovative idea is feasible. In the future, we will let the protein GLBP-vp7-vp1-GFP into Lactobacilli Bifidobacteria, made of an oral bivalent vaccine. It not only can improve the vaccination rate but also does not need to be vaccinated professionals, which the consumption of social resources is lower.
