Genomic Characterization Analysis And Construction Of Saccharomyces Cerevisiae Displaying Cap Protein Of Porcine Astrovirus TJ Isolate

Porcine astroviruses(PAstVs),which belong to the genus Mamastrovirus,are non-enveloped,single-stranded positive-sense RNA viruses that are important pathogens causing intestinal diseases such as diarrhea,dehydration and loss of appetite in piglets.The ability of recombination and cross-species transmission and adaption to new hosts has made astrovirus a potential zoonotic pathogen.Understanding the genetic evolution of astrovirus and developing a safe and effective vaccine,which is one of the most effective ways to prevent the astrovirus infection.The main results are as shown below:1.Genomic characterization analysis of porcine astrovirus T J isolate: In this study,a total of 29 diarrhea stool samples from two regions in Tianjin were examined.The total positive detection rate of astroviruses infection was 13.8 %(4 out of 29 stool samples).PAstV2 and PAstV4 were detected in 3 and 1 of 12 positive samples,respectively.A novel type 4 porcine astrovirus strain(designated as PAstV4/Tianjin/2018)was identified and its complete genomic sequence was determined by RT-PCR and RACE.Sequence analysis showed that PAstV4/Tianjin/2018 has the typical genomic characteristics of an astrovirus,a typical ribosomal frameshifting signal and a conserved subgenomic promoter sequence loated at the overlapping of ORF1a/1b and ORF1ab/2.Genetic evolution analysis indicated that PAstV4/Tianjin/2018 separated into the PAstV4 cluster,together with WBAst V-1/HUN/2011.Recombination analysis indicated that PAstV4/Tianjin/2018 was a novel recombinant strain of strains PAstV4/USA and PAstV4/JPN,and a recombination breakpoint was identified near the ORF1ab/ORF2 junction.2.Construction of Saccharomyces Cerevisiae strain regulated by two prometers and displaying Aga1 efficiently: The laboratory-preserved strain JDY52 was used as the original strain,and the linearized plasmid PIU211 was transferred to JDY52 by Li Ac transformation.Positive transformants were identified by SD-URA solid medium and genomic PCR to obtain a yeast host strain ST1814A which is galactose promoter LAC-regulated and surface-efficiently displaying Aga1.The GPD promoter was cloned by PCR,and the PIU211 Lac promoter was replaced with the GPD promoter by reverse PCR amplification and ligation.The yeast host strain ST1814G,which is GPD-regulated by the glucose promoter and exhibits Aga1 surface-efficiently,was obtained.3.Construction of Cap protein displaying on surface of Saccharomyces Cerevisiae: The PAstV4/Tianjin/2018 Cap protein was used as the surface display protein of Saccharomyces Cerevisiae.The Aga2-Cap transcription units(GAL1-Aga2-Cap-ADH1,GPD-Aga2-Cap-ADH1)regulated by the GAL1 promoter and the GPD promoter were constructed using the Yeast Fab assembly method.The Aga2-Cap metabolic pathway(URR1-TU-LEU2-URR2)was integrated into the host ST1814A and ST1814G ? chromosome HO locus(URR1-HIS3-URR2)by the Li Ac transformation method.Positive recombinants were identified by SD-LEU solid medium and genomic PCR to obtain Cap protein displaying on surface of Saccharomyces Cerevisiae ST1814G/Cap and ST1814A/Cap.4.Analysis of expression characteristics and immunogenicity of recombinant Saccharomyces cerevisiae strains: The expression of Cap protein was analyzed by Western-blot and immunofluorescence.The results showed that Cap protein was expressed and existed on the surface of yeast cells.The serum IgG and fecal IgA levels of the mice after immunization were measured by ELISA,and the results showed that the anti-Cap IgG and anti-Cap IgA titers in the serum of the experimental group were significantly increased compared with the control group.In summary,the whole genomic sequence of the PAstV4 Tianjin isolate was successfully cloned in this study,and its nucleotide sequence and genetic evolution characteristics were determined.Saccharomyces cerevisiae host strains with different promoter-regulated,surface-efficient displaying Aga1 were constructed,which can be used as expression and delivery tools for protein peptides.Cap protein displaying on surface of Saccharomyces Cerevisiae was constructed,which provided a tool for the immune control of porcine astrovirus.