Construction And Immunological Experiment Study Of Virus Like Particles Of RVFV

Rift Valley fever virus(RVFV) is a popular arbovirus zoonosis in the Arabian Peninsula, the African continent and several islands of the Indian Ocean located to the southeast of Africa. In these areas, the virus causes recurrent outbreaks among animals and humans. Domesticated ruminants, particularly sheep, are the most susceptible to the disease. Pregnant ewes after infection can lead to almost 100% of abortion and mortality ratios in newborn lambs can approach 100%. Cattle, goats and ruminant wildlife species are somewhat less susceptible, but losses among these herds can also be considerable. Although most human infections are benign, the virus is risky for its ability to cause hemorrhagic fever and encephalitis. RVFV has been isolated from over 30 mosquito species and several of which have a global distribution. Collectively, these features can explain why RVFV is considered one of the most serious arbovirus threats to human and animal health. The world organization for animal health(OIE) classified it as Class A disease.The Centers for Disease Control and Prevention and the Department of Agriculture in the United States have listed it asClass A pathogen. Moreover, the World Organization for Animal Health has categorized RVF as a Class A statutory reporting animal disease. In China, RVF has been included in the list of 13 exotic animal diseases under priority prevention by the National Medium-Long-Term Plan for Animal Disease Prevention and Control(2012–2020).At present, vaccination is the primary means of preventing injection of RVF. However, On the market there is neither effective anti-RVF drugs nor commercially available RVF vaccine for human use on the market. Vaccines for animals are mainly in the following three kinds: recombinant live carrier vaccine, inactivated vaccines, live attenuated. However, these vaccines have their own shortcomings, and they do not apply to humans. In addition, RVFV can be spread by contact with infectious materials, processing and mosquito bites. At the same time, the two factors of international communication and the ecological environment also seriously affect the spread of the disease and the outbreak. Therefore, the new type of efficient RVFV vaccine is one of the effective means to t be developed. In recent years, Virus-like particles(VLPs) has already showed a huge research value for new vaccines and the potential application as it has the advantages of good strong immunogenicity and safety of explorative.Objective: In this study, RVFV standard strain ZH501 served as the research object, to avoid biological safety problems of the existence of neutralization test agent using RVFV virus, we explored the establishment of neutralization test method for RVFV. We used baculovirus insect cell expression system to construct RVFV virus like particles with stable morphology and high yield, then to evaluate the immune response induced by these particles in vivo. This study could provide some preliminary research and development foundation for the development of virus-like particles prepared in technology-based immunogenic, safe, efficient new RVF vaccines and escort for the healthy development of animal husbandry and human health.Methods:(1) using RVFV structural protein(Gn, Gc) and HIV lentiviral vectors pseudotyped packaging system constructed RVFV pseudotype virus. Electron microscopy and Western blotting were used to identify the pseudotype virus with HIV similarity and detect whether RVFV antibody can inhibit RVFV pseudotyped virus infectionwith the establishment of detecting RVFV antibody neutralizing test method;(2) using the pET-30 a prokaryotic expression vectorto construct containing the truncated Gn protein fragment of recombinant plasmidand transformed into BL21(DE3) competent cells. The expression of the target protein was by IPTG induction. SDS-PAGE and Western blotting were used to detect the expression of protein is correct or not.The expression protein was puried through his label nickel column. The purified Gn RVFV protein immunized mice were detected by ELISA. The specificity of the polyclonal antibody against G protein was detected by Western Blotting;(3)Using pFastBacTMDual vector to construct a recombinant plasmid containing M and S gene, the recombinant plasmid was transformed into Bacmid and the recombinant pFastBacTMDual-M-S DH10 Bac was obtained. Recombinant bacmid pFastBacTMDual-M-S transfected into Sf9 insect cells and continuous blind pass three generations, the recombinant baculovirus rBac-N-G, and the recombinant virus were identified, including extraction of genomic PCR, indirect immunofluorescence, Western blotting and electron microscopy and immunoelectron microscopy. Identification of recombinant baculovirus amplification,-80 C preservation packaging;(4)The correct identification of the 3rd generation or 4 generation of recombinant baculovirus rBac-N-G infected Sf9 cells, via suspension culture for the large-scale preparation of RVFV VLP and centrifuged to remove cellular debris, after ultracentrifugation concentration, sucrose gradient purification, immune indices by intramuscular injection were used to immunize BALB/c mice and horses, detection of mouse antibody levels and IFN-γ, IL-4 and cytokine lymphocyte proportion and number of horse antibody level study its immunogenicity.Results:(1) Observation results by electron microscopy and Western blotting show that package of pseudotype virus are highly similar in size, structure and shape with HIV and infection inhibits test showed that RVFV antibody can inhibit the RVFV pseudotyped virus’ s ability to infect, and the detecting RVFV antibody neutralizing test method is established.(2) The expression of the protein was detected by SDS-PAGE and Blotting Western and detection showed that the target protein was successfully expressed. Purified Gn RVFV protein can induce the mice to produce high levels of Gn polyclonal antibody, and the polyclonal antibody against G protein has a strong specificity;(3) The results showed that the recombinant baculovirus(rBac-N-G) expressing RVFV structural proteins(Gn, Gc and N) were successfully constructed, and the proteins expressed by rBac-N-G infected insect cells Sf9 were self-assembled into VLPs RVFV. The RVFV Gn 、Gc, and N proteins were expressed correctly by indirect immunofluorescence and blotting Western test. The results showed that the virus like particles formed by electron microscopy were in agreement with RVFV in morphology and size;(4) The results showed that the RVFV virus like particles can effectively stimulate mice to produce high levels of neutralizing antibody and improve the proportion and quantity of IFN-γ and IL-4 cytokines in lymphocytes; at the same time, VLPs can also stimulate the horse to produce VLPs high levels of neutralizing antibodies.Conclusion:(1) The RVFV was constructed successfully by using the HIV lentiviral packaging system and the structural protein of RVFV, and the neutralization test method based on RVFV was established, which can effectively evaluate the neutralizing effect of antibody;(2)The Gn protein E. coli BL21 which couldexpress the truncated of RVFV was constructed by using PET-30 a vector and the prepared polyclonal antibody had specificity to full-length G proteinby Western blotting analysis.. The purified protein was immunized to Balb/c mice to prepare polyclonal antibody, which was detected by ELISA, and the titer of the antibody was higher;(3) Using the baculovirus insect cell expression system, the success of expressed RVFV virus like particles; by indirect immunofluorescence and Western blotting identified RVFV gn and GC and N protein expressed correctly and detected by electron microscopy and immunoelectron microscopy. It was found that the formation of virus like particles in the size, shape and typical RVFV virus signatures consistent; by mass culture and discontinuous sucrose density titer centrifugation the RVFV virus like particles;(4)The obtained RVFV virus like particles had strong immunogenicity. RVFV virus like particles can stimulated mouse spleen cells produced high levels of cytokine IL-4 and IFN-γ, and lymphocyte proportion and the number of these two cytokines increased significantly, and induced the humoral immune and cellular immune responses as well as stimulated the mice and horses to produce higher levels of antibodies.