EV71 is a neurotropic virus that often causes clinical symptoms such as fever, hand-foot-mouth disease, and herpetic angina in children. Severe cases can lead to neurological diseases and heart disease, and even death. RV mainly causes viral diarrhea in children. Every season, infants and young children are vulnerable to infectious diseases caused by these two viruses. At present, the coverage coverage of EV71 vaccine is relatively low in the market, and the number of vaccinations is relatively limited. It still takes some time to evaluate the safety and immune persistence of this kind of vaccine. At present, there is no effective treatment for diarrhea caused by rotavirus. The rotavirus vaccines on the market are attenuated rotavirus vaccines, which play a certain role in preventing RV infection, but there are also problems such as intussusception, virulence reversion, and contamination by exogenous factors. Studies have shown many similarities between EV71 and RV viruses. Therefore, we expressed the antigen vp7 of RV virus in tandem with the antigen vp1 of EV71 virus to effectively protect the health of infants and young children. More importantly, compared with injection, oral vaccination is much more convenient, faster to popularize, and does not require professionals to vaccinate, and consumes less social resources.
VP1 is the major antigenic gene of the EV71 virus, which encodes coat protein and promotes infection of host cells by viral particles. Therefore, VP1 was identified as the target gene for the development of an EV71 vaccine. VP7 encodes an important protein of the RV viral coat (the protein of the glycoprotein), which is the primary antigen-activated by antibodies and plays an important role in vaccine development. Therefore, we want to fuse VP1 and VP7 and design the plasmid map of pET28a-VP7-VP1.
Figure 1. Plasmid map of pET28a-vp7-vp1
To build the plasmid, we let the synthetic company synthesize the DNA fragment of VP7 and VP1, and integrate it into the pUC57 vector. Then, we did PCR to amplify the DNA fragment of VP7 and VP1.
Figure 2. The PCR result of VP7 and VP1. The DNA gel electrophoresis image above showed the bands of VP 1 and VP 7 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 located in the correct range within 500 and 1000 bp. Figure 2 proved that we are obtained fragments VP1 and VP7 successfully.
To link the genes of two different viruses vp1 and vp7 together, we connected them by overlap PCR.
Figure 3. The PCR result of VP7-VP1 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. The results showed that vp1-vp7 sequences were successfully fused (Figure 3).
The fusion fragment VP7-VP1 was inserted into the pET28a vector digested with EcoRI and XhoI and transformed it into E. coli DH5α.
Figure 4: 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-vp1were successful (Figure 4).
We send the plasmid pET28a-vp7-vp1 to biological company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region(Figure 5)
Figure 5. Sanger sequencing of pET28a-vp7-vp1 plasmid
Next, the plasmid pET28a-vp7-vp1 was extracted from E.coil DH5α, then transformed into E.coil BL21(DE3) to conduct IPTG induced protein expression.
Figure 6: The PCR identification result of pET28a-vp7-vp1 in E.coil BL21 (DE3)
Out of the 8 line, only 6 and 8 are successful since only their bands are located at around 1700bps. It means that only samples from colonies line 6 and 8 contain proper vp7-vp1 gene fragments. Figure 6 showed that the plasmid pET28a-vp7-vp1 was successful conversion.
We 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 vp7-vp1. Next, we used nickel column purification to purify the protein vp7-vp1 we wanted.
Figure 7. The SDS-PAGE result of the protein vp7-vp1
Referring to the marker in Figure 7, we found the proteins vp7-vp1 in unpurified protein samples (supernatant) and purified protein samples, indicating that proteins were successfully expressed in E. coli BL21 (DE3).
Our experimental results showed that the pET28a-vp7-vp1 plasmid was successfully constructed, the protein was successfully expressed. It proves that it is feasible to express VR-vp7 in tandem with EV71-vp1. We fused two kinds of VR-vp7 and EV71-vp1 expression, not only can avoid the infant by two kinds of virus damage, but also can improve the infant vaccination rate, because oral way was more convenient. In the future, we will fuse the membrane-bound protein GLBP to display on the surface of Bifidobacterium, express the vaccine adjuvant IL-12, and finally, add the green fluorescent protein (GFP) tag to facilitate the detection of vp7-vp1 on Bifidobacterium. Eventually, Bifidobacterium was transferred to make a vaccine that can be taken orally. This method can not only improve the vaccination rate but also facilitate vaccination. In the future, we hope that it can be truly put into use to effectively protect the health of infants and young children.
