Expression And Immunology Study Of E2/Erns Antigen Of Classical Swine Fever Virus In Plant Bioreactors

Classical swine fever virus(CSFV)is a small enveloped virus classified as a member of the Pestivirus genus of the family of Flaviviridae.CSFV infection results in a highly contagious and severe disease,which is characterized by fever and hemorrhage and can run an acute or chronic course.The positive stranded RNA genome contains a single long open reading frame(ORF)that encodes a polyprotein and ultimately yields all final viral proteins through cleavage processing by cellular and viral proteases.At present,two kinds of prevention and control methods are applied,one is large-scale culling used to control the outbreaks in domestic pigs and wild boar and the other is large-scale vaccination used to limit disease spread among domestic pigs and wild boar.Recently,the significant advantages of edible vaccine obtained from transgenic plant,like production and usage,have aroused more and more attention.Structural proteins of CSFV include a nucleocapsid protein C and three envelope glycoproteins,Erns,E1,and E2.The studies on Erns protein have found that Erns protein can also stimulate the production of neutralizing antibodies,which can make the immunized pigs acquire the protective power against CSFV.E2 is the main target of the humoral immune response to CSFV infection.Both E2 and Erns are capable of inducing the production of neutralizing antibodies in the host.In the study,surface antigenic protein of CSFV was expressed by Arabidopsis thaliana bioreactor and Camelina sativa bioreactor,respectively;moreover,the immune effects of expressed protein were also verified.Research results are as follows:1.The optimizations of E2 was carried out according to the codon bias of Arabidopsis thaliana and then the optimized E2 was cloned into plant expression vectors.Plant expression vectors,namely pPHAP1301-E2,was constructed,in which E2 was driven by the promoter of PhaP of Phaseolus vulgaris.2.The optimizations of E2 and Erns were carried out according to the codon bias of Arabidopsis thaliana and then the optimized E2-Erns were cloned into plant expression vectors.Plant expression vectors,namely PHAP1301-E2-Erns,was constructed,in which E2-Erns was driven by the promoter of PhaP of Phaseolus vulgaris.3.The exogenous gene was transferred into Arabidopsis thaliana bioreactor by Floral-dip method,and transgenic plant was obtained by Basta resistibility.It was confirmed that E2 protein and E2-Erns fusion protein,named A-E2 and A-E2-Erns,were expressed by Arabidopsis thaliana after the test of target protein by SDS-PAGE and Western-blot.Oral immunization experiment of expressed A-E2 and A-E2-Erns protein was conducted in mice,which induced greater titer serum IgG,and IgA antibody was also found in the feces of mice.The results showed that the expressed A-E2 protein and A-E2-Erns protein had better immunogenicity.4.Considering the edibility of plant vaccine,Camelina sativa was used in the study as plant bioreactor to express E2 protein and Erns protein of CSFV surface antigen.E2 and E2-Erns were transferred into Camelina sativa bioreactor by Floral-dip method,and total protein of transgenic Camelina sativa seeds was extracted.It was confirmed that E2 protein and E2-Erns fusion protein,named C-E2 and C-E2-Erns,were expressed by Camelina sativa after the test of target protein by SDS-PAGE and Western-blot.Oral immunization experiment of expressed C-E2 and C-E2-Erns protein was conducted in mice,which induced greater titer serum IgG,and IgA antibody was also found in the feces of mice.Oral immunization experiment of expressed C-E2 protein and C-E2-Erns protein were conducted in rabbits.All immunized rabbits can resist challenge infection.The above results explained that the expressed C-E2 protein and C-E2-Erns protein had better immunogenicity,which could be selected as candidate vaccine strain of CSFV plant edible vaccine.CSFV surface antigen was successfully expressed by plant bioreactor in the study,and it was also verified that the expressed antigenic protein had better oral immunogenicity.These results provide supports for production of protein subunit vaccine via plant bioreactor,especially offered the important theoretical basis for the development and application of vaccine candidates for CSFV.