Our experiments could be grouped into two categories based on their purposes: plasmid construction and protein expression. Since we are designing a bivalent vaccine, the most important goal is to be able to express proteins of both VP7 and VP1 on one plasmid. By constructing a plasmid with vp7-vp1, GLBP-vp7-vp1-GFP, and GLBP-vp7-vp1-IL12-GFP genes, we obtained that these genes linked together on a vector. And by showing that proteins are successfully expressed, we are able to prove that the proteins of the genes can be simultaneously coded in a strand of DNA, therefore, confirming that there is potential that a bivalent vaccine can be developed for EV71 and RV.
Figure 1: Gel electrophoresis result showed size of vp1 and vp7 gene fragments. The DNA gel electrophoresis image above showed the bands of vp1 and vp7 genes. In theory, the vp1 gene is 891bp in length, and the vp7 gene is 843bp. The strongly visible bands of cloned vp1 and vp7 genes are located in the correct range within 500 and 1000 bp. The gel electrophoresis helps to examine that we have cloned our genes without error.
Figure 2: Gel electrophoresis result showed vp1, vp7, GLBP, GFP and IL-12 fragments The vp1, vp7, GLBP, GFP and IL-12 DNA fragments PCR result shown in Figure 2. Compared to DNA maker, the PCR products exhibits the correct bands.
Figure 3: Gel electrophoresis result showed length of combined vp7-vp1. This image demonstrated the result of the overlap PCR, in which the purpose is to link the genes of two different viruses together. The length of vp7-vp1 gene is 1734bp. The bands showed the length of the cloned genes 1, 2, 3, 4, 7, and 8 are in the right location relative to the marker. The result showed that even though it was unsuccessful in two scenarios, the rest produced the desired result of linked vp7 and vp1 genes.
Figure 4: Gel electrophoresis result showed overlap extension PCR. Due to large GLBP-VP7-VP1-GFP DNA fragment, we used overlap extension PCR in two steps. Firstly, we splice GLBP and VP7, VP1 and GFP, respectively (shown as left panel). Then, combine two fragments GLBP-VP7 with VP1-GFP to the final DNA fragment (shown as middle panel). The right panel showed the PCR-amplified GLBP-VP7-VP1-IL12-GFP.
Figure 5: Gel electrophoresis result exhibited difference between undigested pET28a and digested pET28a.
To obtain recombinant plasmid, we used restriction enzymes EcoRI and XhoI to digest the gene fragments and plasmid to make double digestion samples, then used DNA ligase to join pET28a vector and vp7-vp1, GLBP-VP7-VP1-GFP, and GLBP-VP7-VP1-IL12-GFP DNA fragment, respectively. After the pET28a plasmids were digested, we used electrophoresis to confirm whether the cleavage was thorough. In vivo, linear (cleaved) plasmids travel a shorter distance compared to circular plasmids due to the more friction. Thus, the digested plasmids in wells 2-4 ran slower than the supercoiled pET28a plasmid in the first well. This result showed that the plasmids were successfully cleaved by restriction enzymes.
The fusion fragment VP7-VP1 was inserted into the pET28a vector digested with EcoRI and XhoI and transformed it into E. coli DH5α.
Figure 6. The colony PCR identification result of pET28a-VP7-VP1/DH5α.
The two lines to the right side of the marker exhibit the bands of the vp7-vp1 in pET28a. The result of the electrophoresis shows a visible band at approximately 1700bp.The PCR identification results showed that the transformation of plasmids pET28a- VP7-VP1 were successful (Figure 6). Thus, we picked up positive colonies to sequence.
Figure 7: Gel electrophoresis result demonstrated the length of pET28a-GLBP-VP7-VP1 -GFP in E.Coli DH10B.
We picked up 10 colonies to verify whether the colony containing the recombinant plasmid pET28a-GLBP- VP7-VP1 -GFP. The result shown as figure 7, and the transformants 2, 4, 7, and 9 have right size. Thus, we picked up positive colony 2 to sequence.
Figure 8: Gel electrophoresis result demonstrated the length of pET28a-GLBP-VP7-VP1-IL12-GFP in E.Coli
DH10B.
We picked up 4 colonies to verify whether the colony containing the recombinant plasmid pET28a-GLBP- VP7-VP1 -IL12-GFP. The result shown as figure 8 and the transformants 1, 3, and 4 have right size. Thus, we picked up positive colony 3 to sequence.
Figure 9. Sanger sequencing of recombinant plasmid The sanger sequencing results showed as Figure 9 and the sequence well matched with the template. Thus, we used this plasmid to perform the subsequent experiment.
BL21 (DE3) is a suitable bacterium to conduct IPTG induced protein expression, so in order for the protein to be successfully expresses in the subsequent experiments, we extracted the recombinant plasmids pET28a-vp7-vp1, pET28a-GLBP-VP7-VP1-GFP, and pET28a-GLBP-RV-EV-GFP from DH5α or DH10B, and transferred them into BL21 (DE3) , added IPTG to induce protein expression, repectively.
Figure 10: Photo of BL21(DE3) colonies grown containing the pET28a vp7-vp1 plasmid.
Figure 11: SDS-PAGE gel demonstrated the size of the protein samples. The negative control is pET28a plasmid without exogenous gene. The expression of VP7-VP1, GLBP-VP7-VP1-GFP, and GLBP-VP7-VP1-IL12-GFP were confirmed by SDS-PAGE, as shown in Figure 11.
Figure 12: SDS-PAGE gel electrophoresis diagram demonstrating the length of the VP7-VP1 protein samples.
We have gained protein from the different concentration of IPTG induced vp7-vp1 BL21 (DE3) bacteria through centrifuge, cell crushing, and purification. The six IPTG concentration as follows: 0.1 mM,0.25 mM, 0.5 mM, 1 mM, 5 mM and 10 mM. In the SDS-PAGE experiment, we took a sample from each of the 6 unpurified proteins (supernatant) and 6 purified proteins. It can be observed that proteins are successfully expressed despite the impurities in the samples.
This image showed the GLBP-VP7-VP1 fused GFP expressed in the bacteria.
Figure13. The comparison of supernatant of GLBP-VP7-VP1 fused 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 14. 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.
Figure 15. The GFP intensity indicated protein expression
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 15, 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.
