Prokaryotic Expression And Immunogenicity Of M-E Protein Of Duck Tembusu Virus AH-F10 Strain

Duck Tubusu virus disease is caused by Duck Tembusu virus(DTMUV),which mainly leads to a decrease in egg production.The incidence of this disease is high.It has been reported in Guangdong,Jiangsu,Anhui and Beijing.The waterfowl has caused serious economic losses.The Duck Tembusu Virus AH-F10 strains was isolated in 2010 years,M,E and M-E gene were obtained by PCR amplification,The results were as follows:516bp,345bp and 879bp.Then cloned into p MD-19-T,plasmid with expression vector p ET-32a were connected,then conversion to BL21 to express the M,E,and M-E protein,expressed mainly in inclusion bodies,the size of each were 40k Da,35k Da and 55k Da.Western blot analysis showed that these three proteins had good reactogenicity.In order to study the immunogenicity of M,E and M-E proteins,healthy and non-immune ducklings were divided into four groups,three groups were immuned M,E and M-E protein 200?g respectively,the last group were immuned PBS 0.2m L.first immune at 14 days,the two weeks later for second immunization,then after two weeks every ducks was challenged with Duck Tembusu virus(10⁴TCID₅₀,TCID₅₀=10^-3.38/0.1m L).The protein immunization effect was evaluated by lymphocyte proliferation test.The protective effect of the protein was evaluated by comprehensive analysis of cytokine detection,specific antibody detection,biopsy and pathology.Lymphocyte proliferation test results showed that group M,E and M-E SI value was significantly higher than group PBS.The group M-E,M,E levels of IL-4 and IFN-?were higher than group PBS.The indirect ELISA results showed that antibody levels in the order group M-E,group M,group E and group PBS.M-E group compared with group PBS the differences was significant at 42 days,49 days and 56days.And M group compared with group PBS the differences was significant in the42 days.Analysis of group PBS found the most serious lesions,including green to gastric cuticle in Stomach.Histopathological findings showed that the tissue lesions in the group PBS were more severe than those in the group M,E and M-E,and the cells were necrotic in the brain,liver,kidney and ovary.In summary,the immune M-E protein,M protein and E protein can produce good immunoprotective effect to Duck Tembusu virus,and ME protein is the best.For the development of Duck Tembusu virus vaccine and disease prevention to provide a basis.