Fusion Expression Of Salmonella Abortus Equi FliC And Equine Herpes Virus GD And The Immunopotentiating Effect Of FliC On Recombinant GD DNA

Salmonella abortus equi could cause abortion of equus animal which also named equine paratyphoid.The typical characteristics of this disease is abortion of pregnant horses.With the increase in the number of horses in Xinjiang this disease has seriously affected the development of the horse breeding industry.Equine rhinopneumonia(ER)is one of the most important infectious diseases caused by the equine herpesvirus 1 and type 4,and equine herpesvirus 1 causes abortion of pregnant mare and serious harm to the horse breeding industry of XinJiang.The wide spreading of ER has posed huge economic loss.FliC is important flagellin and antigenic components of Salmonella abortus equi.Glycoprotein D is effective proteins for the protection and prevention against equine herpesvirus.In order to investigate the molecular adjuvant effect of flagellin FliC on equine herpesvirus gD.The fusion expression of Salmonella abortus equi FliC gene and equine herpesvirus gD gene and the immunopotentiating effect of FliC on gD DNA was elucidated in this study.In this study,The FliC gene was amplified by PCR from genomic DNA of Salmonella abortus equi and gD gene of equine herpes virus was amplified by PCR.The fusion gene FliC-gD were constructed by overlap PCR technique and inserted into pVAX1 vector.The expression of FliC,gD and FliC-gD proteins were identified by indirect immunofluorecence assay and Western-blotting assay.These recombinant plasmids were used to immunize 5 groups of C57BL/6 mice,pVAX1-gD,pVAX1-FliC,pVAX1 and PBS were used as control group.Three weeks after second immunization,mice were infected with 1×10~5 PFU of EHV-1-XJ2015 and the mice were challenged with the minimum lethal dose of Salmonella abortus equi2×10~9 CFU/ml.Serum of immunized mice were collected and Anti FliC/gD specific antibody,antibody subtype,IFN-?and IL-4 level were detected,the lungs were used to virus detection and histopathological observation.The results showed that the eukaryotic recombinant plasmids pVAX1-FliC,pVAX1-gD and pVAX1-FliC-gD were constructed.IFA and western-blotting result indicated that recombinant plasmids could be expressed in vitro and the purified recombinant plasmid were used to immunized mice.After immunization,the antibody titer of mice antisera could reach to 1:3200,and the levels of IgG1,IgG2a,IFN-and IL-4 were significantly higher in the serum of mice immunized with expression plasmid pVAX1-FliC-gD than pVAX1-FliC and pVAX1-gD immunization mice(P<0.01).The protective effect of pVAX1-FliC-gD immunized group could reach to 58.3%after challenging with Salmonella abortus equi in mice.The virus DNA amount of mouse lung was detected by realtime PCR.The results showed that the virus DNA amount of pVAX1 and PBS immunized group was the highest at the 5th day after challenge,reach to 3.3×10~4 copies/?L.The virus DNA amount of pVAX1-FliC-gD immunized group and pVAX1-gD immunized group significantly induced than pVAX1 and PBS immunized group(P<0.01).The histopathological observation result showed that pVAX1-FliC-gD immunization group pVAX1-gD immunization group could effectively reduce the reproduction of equine herpes virus in the lung and the pathological changes of lung.These results indicated that FliC was a better molecular adjuvant could significantly enhanced the immune effect of the equine herpesvirus gD.This study which laied the foundation for the development of new type of equine herpesvirus virus vaccine.