| Zika virus is a yellow virus transmitted by Aedes mosquitoes,which can cause microcephaly of newborn in pregnant women.Research have confirmed that Zika virus infection is one of the causes of Guillain-Barre syndrome;due to the outbreak of the ZIKV epidemic in countries such as Vietnam that borders China,China is faced with potential input risks.Because virus-like particle(VLP)are similar in structure to virus particles and contain no virus genetic material,they are highly immunogenic and can induce organisms to produce high-titer antibodies.This is the safest and most effective vaccine development strategy.In this study,the Pichia pastoris expression system was used to express recombinant ZIKV envelope protein(Env).After the recombinant protein was purified,VLP were assembled in vitro.Animal experiments were performed to evaluate the protecting efficacy of vaccine candidate.Objective:In this study,the ZIKV envelope protein was used as a research object and obtain ZIKV envelope protein by the Pichia pastoris expression system.A mouse infection model was established that evaluate VLP vaccine protection,which provides a pre-development basis for the development and preparation of a virus-like particle-based,immunogenic,safe,and efficient Zika virus vaccine.Methods:(1)Construction of recombinant expression vector,expression of ZIKV envelope protein by Pichia pastoris expression system,selection of high copy strains by different concentrations of zeocin antibiotics.(2)Induction of expression of target protein,first purification of recombined target protein by nickel ion chromatography and then further purified by molecular sieve chromatography.The identification of target protein expressed successfully by immunoblotting and mass spectrometry.(3)According to different ionic strength and pH value of the assembly buffer,the optimal conditions in vitro assembly of VLP were selected.Dynamic light scattering and transmission electron microscopy were used to identify the assembly effects of VLP.(4)The mice immunized with high purity ZIKV VLP,that were assayed by ELISA and cell-neutralization experiments to determine the level of humoral immunity induced by the change of antibody titer in the body of mice.The detection of IFN-y and IL-4 cytokines secreted of memory lymphocytes by elispot and intracellular cytokines,which evaluated the ability of secrete INF-y and IL-4 by memory CD8+T and CD4+ T cells,to evaluate their cellular immunity.A mouse infection model was established,and challenge experiments were conducted to verify the protective ability of the candidate strain of ZIKV VLP vaccine for mice.RESULTS:(1)The recombinant vector constructed was verified by PCR and sequencing of the bacterial liquid.The results showed that the recombinant plasmid was constructed correctly.(2)The successful expression of ZIKV Env was confirmed by Western Blot and protein spectroscopy.(3)The assembly effect was identified by dynamic light scattering and transmission electron microscopy.It was proved that the assembled VLP was spherical and basically the same size as the natural ZIKV particle.ZIKV VLP was successfully assembled in vitro.(4)Plaque reduction neutralization test demonstrated that ZIKV VLP can induce the generation of high titer neutralizing antibodies,elispot and intracellular cytokine staining assays demonstrating that a strong cellular immune response can be induced.(5)Mouse animal challenge protection experiment demonstrated that the ZIKV VLP can completely protect the immunodeficient mice(A129)from lethal dose of virus attack.Conclusions:ZIKV Env was successfully expressed in Pichia pastoris expression system,ZIKV VLP immunizes immunodeficient mice(A 129),which protects mice against lethal doses of virus challenge.The candidate strain of the ZIKV VLP vaccine obtained in this study is expected to be developed as a safe and effective ZIKV vaccine.In addition,here,we develop an ultrasensitive and instrument-free EBOV detection assay based on colloidal carbon immunochromatography.Carbon nanoparticle-labeled rabbit anti-EBOV-VP40 IgG were concentrated in the conjugate pad and monoclonal antibody(McAb,4B7F9)against EBOV-VP40 and goat anti-rabbit IgG were immobilized on the nitrocellulose membrane with 2μL/cm at a concentration of 1 mg/mL as test and control lines,respectively.Then the sample application pad,conjugate release pad,nitrocellulose membrane and absorbent pad were assembled into a lateral flow test strip.The test strip shows strong specificity against related viruses that share similar clinical symptoms and geographic range with EBOV,including Marburg virus,influenza virus,yellow fever virus and dengue virus.Furthermore,the strip in this study could be as point of care(POC),ultra-sensitive and specific promising candidate for EBOV serological screening in rural Africa or entry/exit ports. |
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