Expression Of Classical Swine Fever Virus E2 Protein In Pichia Pastoris And Preliminary Study On Immunological Activity

Classical Swine Fever(CSF)is a highly contagious hemorrhagic disease affecting pigs.E2 protein is an important structural protein of CSFV and the main protective antigen of CSFV.The expression system of pichia pastoris is characterized by secretory expression and eukaryotic embellishment and it is the optimal expression system for E2 p rotein.E2 protein is an ideal candidate antigen for the development of subunit vaccine and relevant detection technology of CSF.In this study,pPICZ?A-E2 vector of pichia pastoris was constructed using the extracellular domain gene of CSF V E2 protein as the template.The positive clones of pichia pastoris X-33 strain were obtained by electrophoresis,and the CSFV E2 protein expression strain of X-33 was identified by SDS-PAGE and western-blot.The optimal expression of E2 protein in shaker was obtained by exploring the pH of induction medium,the concentration of inducer and the induction time.The E2 protein was purified by Ni NTA affinity chromatography,ion exchange and other purification methods,and its glycosylation modification and dimer structure were analyzed.The purified E2 protein was immunized to animals,and the immunogenicity of the protein was analyzed according to serological test.The results showed that the E2 protein of CSFV was successfully expressed in pichia pastoris and is a dimer structure with glycosylation modification.The results of animal experiments showed that the protein had good immunogenicity.This study lays a foundation for the research of subunit vaccine of CSFV,monoclonal antibody screening and the development of relevant detection technology.The overall implementation plan is mainly divided into the following parts:1.pPICZ?A-E2 vector constructionAccording to the E2 amino acid sequence(GenBank: AF531433)of rabbit attenuated vaccine E2 protein of CSFV in NCBI database,the extracellular gene of E2 sequence(345 aa)was amplified and insert into pPICZ?A vector.PCR identification and gene sequencing showed that the recombinant vector was successfully constructed.2.Electric transformation and monoclone screening of yeastThe extracted plasmids linearized by PmeI could be used for electroporation with a mass of 6.14 ?g determined by uv spectrophotometer.The results showed that all the cloned were positive identified by PCR.3.Expression and purification of CSFV E2 proteinInducing the positive X-33 strains identificated by PCR with 1 methano l %.Collecting the medium supernatan 1 mL on the third day,enriched by Ni over night at 4 °C.Western-blot analysis showed that the recombinant E2 protein was successfully expressed.Different pH induction medium,different induction time and different concentration of inducer were set up.The highest protein expression was obtained when induction time was 5 days,concentration of inducer was 0.5%,and pH of medium was 3 or 4.The concentration of impurities and eluents in protein purification was determined,and the best purification method was determined.4.Analysis of E2 protein activityThe activity of E2 protein was detected by dot blot assay,which was improved from Sithigorngul(1991).Taking a piece of nitrocellulose membrane(NC)membrane,each point with samples was loaded 1 ?L,drying the membrane at 60 °C,then soaked in PBST contains 5 % skimmed milk,and blocked for 2 h at room temperature.Washing the nitrocellulose membrane with double distilled water for three times.Incubating the nitrocellulose membrane with corresponding primary antibodies and secondary antibodies,with ECL developing.5.Identification of protein structure10 ?g purified E2 protein was treated with N-glycosidase F(PNGase-F).The purified protein was subjected to non-ruducing treatment and the protein dimer structure was analyzed by western blot.The results showed that E2 protein was glycosylated and existed as dimer.6.Identification of protein structureThe purified E2 protein was mixed with freund's adjuvant at 1:1 ratio and emulsified as an immunogen for the primary immuning.E2 protein was emulsified with freund's incomplete adjuvant at 1:1 ratio as a secondary immunogen.Immuning the rabbits and mice respectively,collected the serum every week and preserved at-20 °C for serological analysis.The blocking rate of rabbit serum against CSFV was 92.5%,and that of mouse serum against CSFV was 58.0%.The IPM A test showed that the mice serum could react with CSFV.The purpose of this study lies in the successful establishment of CSFV recombinant E2 protein yeast expression system,we obtained high pure E2 protein and analyzed and identified its glycosylation embellishment and dimer,and evaluated its immune activity.These studies showed that the immunogenicity of CSFV recombinant E2 protein expressed by pichia pastoris was great,providing a certain bas is for monoclonal antibody screening,the development of new subunit vaccine and the development of relevant detection technology.