Development Of Virus-Like Particles Vaccine Against Porcine Circovirus Type 3 By Saccharomyces Cerevisiae

Porcine circovirus type 3 is a kind of circovirus reported from the United States in 2016,which is associated with porcine dermatitis and nephrotic syndrome,reproductive disorders and multi-system inflammation,causing significant losses to the global pig industry.Due to the widely spreading of virus,developing effective and safe vaccines against PCV3 is rgently needed.At present,there is no report about the isolation and culture of PCV3 virus at China and abroad,which restricts the preparation of attenuated and inactivated vaccines.Based on existing characters of CAP protein,self-assembled into Virus-like particles(VLPs)in vitro,we construct the secreted virus like granule vaccine of PCV3 by Saccharomyces cerevisiae in this study.The content of specific antibody in the serum of immunized animals increased,which indicated that the vaccine could activate the immune response successfully,so it has the prospect of further application and development.The main results were listed as follows:1.Construction of secretory recombinant Saccharomyces cerevisiae expressing PCV3 CAP protein.With Yeast Fab assembly method,the complete transcriptional units containing promoter(GPD),target gene(CAP)and Terminator(ADH1)was successfully constructed using the characteristics of type II restriction endonuclease,then inserted them into the Ⅳ chromosome of yeast by homologous recombination.The positive transformants were screened by leucine deletion plate,and the recombinant Saccharomyces cerevisiae strain expressing PCV3 CAP protein was obtained ST1804G-01.2.Kinetic analysis of CAP protein expression in recombinant yeast and electron microscopic observation of VLPs.Western blot showed that the recombinant yeast could stably express PCV3 CAP protein.The yield of PCV3-CAP protein was detected at 12 h,24h,36 h and 48 h respectively.The highest yield was 1.81mg/L at 36 h.The assembly of PCV3 VLPs was detected by transmission electron microscopy.The regular VLPs structure with diameter of 18 nm was found in the concentrated yeast supernatant,which was consistent with the expected size.3.Evaluation of the immune effect of oral PCV3 VLP vaccine.Twenty-four three-month-old piglets were randomly divided into two groups.They were immunized orally on days 0,4,7,14 and 21.The experimental group was immunized orally with 20 m L PCV3 recombinant Saccharomyces cerevisiae strain(ST1804G-01)and the control group was immunized orally with original Saccharomyces cerevisiae strain(JDY52).Blood samples were collected on day 24 to detect the level of specific antibody in serum.Compared with the control group fed with yeast,the specific IgA and IgG in the serum of the experimental group increased significantly.4.Construction of PCV2 and PCV3 multivalent recombinant yeast strains.Circovirus type 2 and 3 are the main virulent strains of porcine circovirus.Using the complete transcriptional units of PCV2-CAP protein which has been constructed in our laboratory,we designed primers to realize the tandem of PCV2-CAP and PCV3-CAP protein transcription units.The recombinant Saccharomyces cerevisiae strain which secreted PCV2 CAP protein and PCV3 CAP protein simultaneously was screened by leucine deletion plate.The two proteins expressed well detected by Western blot.In conclusion,the recombinant Saccharomyces cerevisiae strain with PCV3-CAP protein secretion and the recombinant yeast strain with PCV2-CAP and PCV3-CAP tandem expression were successfully constructed in this study,which provided an effective strategy for the immune control of porcine circovirus.