Research On Surface Displayed Saccharomyces Cerevisiae Vector Of Zika Virus Vaccine

Saccharomyces cerevisiae（S.cerevisiae）is a unicellular eukaryotic microorganism,which is widely used in food industry,so it is considered to be safe.With the development of molecular genetics and molecular biology technology,its genome sequence has been completely measured.Many expression vectors that can be applied to S.cerevisiae have been developed.Zika virus（ZIKV）belongs to the Flaviviridae family,genus Flavivirus,and is an arbovirus.ZIKV has the characteristics of fast speed and wide spread.Therefore,developing a safe and effective vaccine is undoubtedly a top priority.Although there is a certain depth of research on the pathogenesis of ZIKV,it is very regrettable that there are no effective antiviral drugs or vaccines for the treatment or prevention of ZIKV infection.There is no doubt that vaccination is one of the most effective measures to prevent ZIKV infection and outbreak.Developing a safe and effective vaccine is the top priority.In this paper,the immunogenic Envelope（envelope）gene on the surface of ZIKV was used as the research object to construct the surface displayed S.cerevisiae EBY100/pYD1-Envelope,and its immunocompetence was analyzed.The purpose was to establish the delivery technology platform of the virus oral vaccine based on the surface displayed S.cerevisiae.（1）A surface displayed S.cerevisiae EBY100/pYD1-Envelope was constructed by conventional molecular biology.Basing the gene of Zika/SZ01/2016 as a template,designing the Nhe I enzyme cutting site in the upstream primers and the EcoR I enzyme cutting site in the downstream primers containing stop codon（TAA）.The Envelope gene amplified by PCR was cut through Nhe I/EcoR I double enzyme,and was connected by the plasmid pYD1 which was also cut through Nhe I/EcoR I double enzyme.Then it was transformed into E.coli DH5αcompetent cells,screening positive clones,extracting its plasmid DNA and naming it as pYD1-Envelope.Then transferring it to EBY100 competent cells,the selected positive S.cerevisiae was named EBY100/pYD1-Envelope.（2）In order to detect the location of Envelope protein in S.cerevisiae,the lysates and supernatants of EBY100/pYD1-Envelope were analyzed by Western blot.The results showed that the expression of Envelope protein was in the EBY100/pYD1-Envelope cells.The surface of EBY100/pYD1-Envelope was treated by polyclonal anti-Envelope sera and analyzed by immunofluorescence and flow cytometry.The results showed that the specific antigen protein existed on the surface of EBY100/pYD1-Envelope.In addition,the content of Envelope antigen protein was determined by BCA protein quantitative kit,and its expression amount was 0.3305μg/OD_600nm.（3）Immunocompetence analysis of surface displayed S.cerevisiae EBY100/pYD1-Envelope.Envelope specific serum IgG,IgM antibody titer and fecal IgA antibody level were detected by ELISA.Investigating Regimen1,Regimen2 and Regimen3 three immunization regimens.The results showed that Regimen3 is the best immunization regimen.Based on the technology of surface displayed S.cerevisiae,EBY100/pYD1-Envelope was successfully constructed,and the surface detection platform for Envelope antigen protein was established.The titer of Envelope specific antibody was detected by ELISA,and an antibody response analysis platform for oral immunization was established.This paper only uses the Envelope genome of ZIKV as a research pivot,and the yeast surface display technology is a common platform,which provides new research strategies and methods for the development of other virus or bacterial vaccines.