| West Nile virus (WNV) belongs to genus Flavivirus within the family Flaviviridae.WNV possesses a single-stranded, positive-sense RNA genome of approximately 11 kb, which contains a long open reading frame coding for a polyprotein precursor. The polyprotein is processed into three mature structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by cellular and viral proteases.WNV can cause aseptic meningitis, lethal encephalitis or flaccid paralysis. WNV was first isolated in 1937 from the blood of a febrile patient from Uganda. Historically, outbreaks of WNV disease occurred in Africa, Europe, Asia, the Middle East, North America and other regions.So far, no specific therapies or vaccines for WNV infection are currently approved for human use. Only the inactivated vaccines were used in equines.The ectodomain of E protein forms three structural domains (I, II, and III). DIII, located on one side of DI, adopts a seven-stranded immunoglobulin (Ig)-like fold and has been implicated in receptor binding. WNV specific antibodies that neutralize infection most efficiently bind to epitopes (E307, E330, E332) on the lateral ridge of DIII.In the study, it was investigated that the effects of the domain III of West Nile virus E protein on viral infection and immunization with the domain III of West Nile virus E protein protects mice against lethal infection with West Nile virus. This work contributes to the understanding of functions of the domain III of West Nile virus E protein and the developement of West Nile virus subunit vaccine in China. Main results are described below.1. Prokaryotic expression and antigenicity identification of the domain III of West Nile virus E proteinThe gene of the domain III of West Nile virus E protein was amplified by RT-PCR from BHK-21 cells infected by West Nile virus AY490240 strain and cloned into prokaryotic expression vector pET32a. After been verified by sequencing, the fusion protein was espressed under IPTG induction in the competent E. coli BL21 (DE3) LysS. The expression product was identified by SDS-PAGE. Ni-NTA colume was used to purify the domain III fusion protein. The domain III fusion protein could be recognized by anti-His-tag monoclonal antibody and rabbit anti-WNV polyclonal antibody with Western blotting and was ready for its function study.2. Influence of the domain III of West Nile virus E protein on West Nile virus infectionTrx-His-S-tag of the domain III fusion protein was removed by cleavage of recombinant Enterokinase. The roles what the domain III of West Nile virus E protein plays in West Nile virus infection were investigated by Plaque reduction assay. In this study we found that the domain III of West Nile virus E protein could inhibit West Nile virus entry in BHK-21. The mechanism of competitive inhibition may play an important role in this process.3. Immunization with the domain III of West Nile virus E protein protects mice against lethal infection with West Nile virusTo observe the immunogenicity of recombinant protein, the pET32-WNEIII was hypodermically injected into Balb/c mouse with Freund's adjuvant. Mouse was vaccinated three times with 2 weeks interval at a dose of 25μg in a volume of 200μl. Specific antibodies against pET32-WNEIII could be detected by IFA 2 weeks after the first injection. Neutralizing antibody responses to WNV was detected by Plaque reduction neutralization assay. The immunoprotection of pET32-WNEIII fusion protein against West Nile virus was observed in mice 2 weeks after the last booster. The antibody titer and specific neutralizing antibody reached to peak after the third injection, respectively 1:2560, and 1:160. The results showed that mice anti-WNV neutralizing antibody was elicted by domain III fusion protein. Compared with the control group, the domain III of West Nile virus E protein could protects 80% mouse against lethal infection with West Nile virus. All the data indicated that the domain III of West Nile virus E protein could provided good protection against WNV challenge in mice.This study exhibited that the domain III of West Nile virus E protein could induce homoral immune responses and provides good protection against WNV challenge in mice. These lay a solid foundation for further developing high titer subunit vaccine of West Nile virus...
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