Preparation Of Porcine Vesicular Disease Virus VLP In Yeast And Preliminary Evaluation Of Its Immunogenicity

Porcine vesicular virus belongs to the genus enterovirus in family Picornaviridae which can cause highly infectious swine vesicular disease with symptoms similar to foot-and-mouth disease.Clinically difficult to differentially diagnose vesicular diseases such as foot-and-mouth disease,because the symptoms of SVDV like blisters and ulceration in the hoof and mouth witch are almost as same as the others.This situation has caused great economic losses and no commercial vaccine is currently available.So it is necessary to study the SVDV vaccine.The appearance of virus-like particle provides a reference for vaccine preparation because of its' structure resembles to the virus capsid structure and they can cause specific immune responses without infecting.The Pichia pastoris expression system has the characteristics of simple operation,cheap consumables,and high expression,and it is effective for expressing foreign proteins.In this study,the Pichia Pink Expression System was used to express the SVDV P1 structural protein precursor gene and 3CD Protease gene to prepare VLP.This will provide a reference for the subsequent preparation of SVDV VLP vaccine.1.Optimized the P1 and 3CD gene sequences according to yeast codon preferences,then compound.Insert the P1 and 3CD genes into pPink-HC respectively to construct pPink-SVDV-P1 and pPink-SVDV-3CD.Use isocaudamer Bgl?&Bam H?to cut 3CD gene and its' gene expression cassette.Insert the fragment into pPink-SVDV-P1 which digested with Bgl?.Recombinant expression plasmid construction completed who named pPink-SVDV.2.The successfully constructed recombinant plasmid was transformed into yeast competent cells by electrotransformation.PAD auxotrophic medium was used to screen white spots.Select high-copy positive transformants,extract yeast DNA after proliferation and use P1 and 3CD gene-specific amplification primers for PCR identification to select the correct recombinant yeast.3.The acid-washed glass beads were used to shake and break the yeast cells to release proteins,which were identified by SDS-PAGE and Western-bloting.The results showed the presence of VP0,VP1,VP3 structural proteins.It is proved that both P1 and 3CD have been successfully expressed and have natural activity.3CD protease breaks down P1 structural protein precursor into 3 single structural proteins.Observation by DLS and TEM identified the existence of round hollow particles with a clear particle size of about 30 nm,demonstrating the successful assembly of structural proteins into VLP.Western-bloting identification proves that VLP can specifically bind to antigen,and initially proves that it has certain immunogenicity.In summary,in this study,we used pPink-HC as a vector,not only successfully constructed a yeast expression recombinant plasmid pPink-SVDV containing structural protein precursor P1 gene and protease 3CD gene,but also successfully expressed P1 and3 CD proteins which have natural activity.The VP0,VP1 and VP3 structural proteins produced by enzymatic hydrolysis were proved to be able to self-assembled into VLP.This study laid the foundation for the study of SVDV's VLP vaccine.