Preparation Of Virus-like Particles For Porcine Circovirus Type 2 By Saccharomyces Cerevisiae

Porcine circovirus type 2?PCV2?has caused great economic losses to the global pig industry.Vaccination is one of the effective measures to control the disease,And the capsid protein subunit vaccine.as the current mainly immune strategy were used in practice.The eukaryotic cell model Saccharomyces cerevisiae is used as the preferred host for gene function digging or exogenous protein expression due to its clear genetic background and maturing engineering method.Based on the characteristics of PCV2structural protein?Capsid?self-assembly into virus-like particles?VLPs?in vitro,the PCV2 Cap protein secreted expression by Saccharomyces cerevisiae in 2 different yeast expression vectors were constructed and the PCV2 VLP were prepared in our study.This study is divided into the following parts:?1?Molecular design of multi-copy expression vectors:Using the Saccharomyces cerevisiae genome delta sequences,the target gene-integrated plasmid with delta homologous arm at both ends was constructed to obtain highly expressed strains.On the basis of this,the Ptpi promoter was replaced with the Gal promoter by using the ClonExpress^TM technique.The PCV2 Cap genetic codon was optimized and Cap-ori/opt were inserted into the expression vector of YCplace33-Ptpi/Gal-xyn2s-MCS-TadhI to explore the effect of different promoters on protein expression.?2?Construction and screening of recombinant Saccharomyces cerevisiae expression vector:We also use a method called YeastFab Assembly to synthesize transcription units in a single tube by synthesizing these genetic elements in Saccharomyces cerevisiae as a standardized biological site.The PCV2-Cap protein gene?Gen Bank accession number:KT261750.1?was optimized by Saccharomyces cerevisiae preference codon.Promoter?PRO?,open reading frame?ORF?and terminator?TER?were loaded using the modified pSMART HCKan plasmid.The type IIs cloning method was used to assemble the transcription unit in a single tube to construct an integrated single copy secretory carrier,through the RFP reporter gene and different resistance screening,the positive rate up to 90%or more.The recombinant plasmids of W303a?YCplac33-Gal-xyn2s–Capo-Tadh I?and JDY52?GPD/TEF2-xyn2s-Capo-ADH1?were successfully constructed by the combination of the two recombinant plasmids into the genomes of Saccharomyces cerevisiae W303a and JDY52.The recombinant plasmids were identified by different antibiotics and PCR,the rate of positive recombinants were screened at 85:1 and 10:1,respectively.?3?Two kinds of recombinant yeast expression and preparation of PCV2 VLP:recombinant yeast were successfully constructed by transformed the two recombinant plasmids into the genomes of Saccharomyces cerevisiae W303a and JDY52.The recombinant Cap protein was demonstrated by SDS-PAGE,Western bloting and transmission electron microscopy.The expression rates of two different expression vectors VLP containing GPD and TEF2 were 12?g/m L,25?g/mL.In this study,a variety of recombinant expression vectors were constructed using different expression elements,and their superiority and disadvantages were explored;and we successfully constructed a recombinant yeast expressing PCV2 Cap protein secretedly;it was further demonstrated by electron micrography that the yeast-derived PCV2 Cap protein could self-assembles into virus-like particles?VLPs?.These results show that it is feasible to use S.cerevisiae as a safe and simple system to produce PCV2 virus-like particles,and that oral yeast-mediated antigen delivery perhaps a potential new PCV2 subunit oral vaccine,which can be used as a potential vaccine to protect pigs from PCV2-infection...