Construction Of Recombinant Baculovirus Expression Vector Of Chimeric Rotavirus And Enterovirus 71 Virus-like Particles

Human rotavirus (HRV) is one of the main pathogen of infantile diarrhea, which infected almost all children under the age of 5 at least once, thus is an important cause of infant death in developing countries. These two kinds of virus serious threats to human health and public safety. There is no effective treatment for these two diseases, and live attenuated and inactivated vaccines were still the main method to prevent and control these two diseases. However, live attenuated vaccines may occur gene recombination and virulence, inactivated vaccines are rarely even unable to induce cellular immunity, and there is a potential risk of not completely inactivated, thus these two vaccines are unable to achieve the ideal effect. Therefore, it is of great significance to develop a novel genetic engineering vaccine against rotavirus and Enterovirus 71. Virus like particles (VLPs) mimic the structure of virus particles, thus can elicit strong humoral immunity and cellular immunity. VLPs completely lack the DNA or RNA genome of the virus and thus without any of the drawbacks of reversion, recombination and re-assortment, and that lower doses of antigen. Therefore, VLPs has become an ideal genetic engineering candidate vaccine in recent years. These findings laid a theoretical foundation for the development of novel chimeric Rotavirus and EV71 virus-like particles vaccine.Previous studies confirmed that co-expression of RV VP2、VP6 and VP7 in Sf9 cells can be assembled into three layers virus like particles, and have a good immune effect. In addition, studies found that VP4 of rotavirus can help VLPs to enter the intestinal cells efficiently, display of EV71 VP1 on the VLPs surface can induce high levels of antibodies and has a good immune protective effect. In this study, PCR was first used to amplify VP2、VP4、VP6、VP7 gene of RV and VP1 gene ofEV71, and then fusion PCR technology was used to amplify ER1,2 fusion gene, which is composed by VP1 gene of EV71 and truncated VP2 gene of RV through the flexible linker. The correct viral structural genes were cloned into transfer vector pFBDM-IM and pUCDM-XIGP and then integrated into Bacmid through Tn7 and Cre-LoxP recombination. Sf9 cells were transfected with recombinant Bacmid to obtain recombinant baculovirus. Sf9 cells were then infected with baculoviruses and observed with fluorescence microscope at 72 hours post infection. There is obvious cytopathic effect, Such as cell significantly larger, cell proliferation was not obvious or not,Cells from the bottle wall fell off and fluorescence in Sf9 cells, which confirmed the successful construction of recombinant baculovirus. The genome DNA was extracted and then identified by PCR with specific primers of EV71 and RV gene. The results showed that the foreign gene had been successfully integrated into baculovirus genome. The expression of exogenous genes were examined by SDS-PAGE and Western blot. The results showed that VP2、VP6、VP7、ER1,2 were expressed correctly as the expected size, respectively, Protein size was 104 KDa、46 KDa、38 KDa、127 KDa. Thus, genes related to virus like particles were expressed correctly, which laid the massy foundation.