New Zika Virus Vaccine Research

Background The Zika virus(ZIKV)was found in the ZIKA forest in Uganda,Africa,and belongs to the Aedes mosquito-bone yellow virus.About one-third of the world’s population was lived in its endemic.area,and the virus is closely related to Guillain-Barre’s syndrome,neonatal microcephaly,and other diseases.Zika virus is a member of the family of Flaviviridae.Its genome is a single-stranded positive RNA of-10.7KB and the virus particles are spherical.E protein is a fusion protein contaiming three domains and plays an important role in virus attachment to cells.Currently,Zika virus vaccine research has varied,with advances in attenuated live vaccines,recombinant subunit vaccines,nucleic acid vaccines based on various vectors,and virus-like particle vaccines.However,these vaccines are all in the preclinical phase and it is particularly important to develop a safe and effective Zika vaccine as soon as possible.Objective Zika virus E protein was expressed in the prokaryotic system to detect its immune response in mice and to detect neutralizing antibodies in mouse serum by plaque assay.Method First of all,prokaryotic expression plasmid pET28a-E was constructed and transformed into E.coli competent BL21(DE3),Optimized protein expression induced concentration and temperature.The protein was purified by Ni-NTA column,and its protein purity was detected by SDS-PAGE and its immunogenicity was detected by western blot The final concentration of Zika virus E protein was determined by the BCA protein quantification kit Secondly,pCAGGS and pcNDA3.1 expression plasmids were used as the expression vectors of Zika virus prME protein,and in order to make prME protein secreted to the extracellular,Japanese meningitis virus signal peptide JEV and human signal peptide TPA were selected as protein secretion.Then,four plasmids pCAGGS-JEV/TPA-prME and pcDNA3.1-JEV/TPA-prME were constructed by enzyme digesrtior:BALB/c mice and ICR mice that were ordered 6-8 weeks were randomly divided into experimental group and control group.Immunize 50 μg of Zika E Protein on Day 0,21 and 35.Blood was collected from the eyelids on days 0,21,35,and 49,and IgG titers were measured by enzyme-linked immunosorbent assay(ELISA).In the Biosafety Laboratory(BSL-2),vero cells were used to amplify live Zika virus,and neutralizing antibody titers in serum were measured by Plaque Reduction Neutralization Test(PRNT).Result The prokaryotic system expression plasmid was successfully constructed,and the optimal Zika virus E protein expression conditions were selected.Zika virus E protein was obtained through nickel column purification.The purified protein-immunized mice can elicit an immune response in vivo and Neutralizing titers can be detected in Zika virus Plaque Reduction’ Neutralization Test.At the same time,four DNA expression plasmids were successfully constructed,and the expression of the plasmid was verified at the cellular level,which provided the basis for further experiments.Conclusion Zika virus E protein can be expressed in the prokaryotic system.After purification,the protein carn be immiunized intraperitoneally to mice,which can induce an immune response in mice and produce neutralizing antibodies against Zika virus.