| Avian hepatitis E virus(a HEV) is a kind of virus associated with hepatitis-splenomegaly syndrome in chickens. a HEV is a member of the genus Hepevirus that also includes human hepatitis E viruses(h HEV) and swine hepatitis E viruses(s HEV). a HEV is a non-enveloped, single stranded positive sense RNA virus. The complete genome of a HEV is 6.6 kb including two noncoding regions and three open reading frames(ORFs). Phylogenetic tree analysis reveals that a HEV represents the fifth genotype that is distinct from h HEV and s HEV. a HEV displays antigen relevance with h HEV, and avian hepatitis can be used as an effective model to study the replication and pathogenesis of h HEV. a HEV is mainly transmitted by the fecal-oral route, and cause the clinical disease in laying and broiler chickens with age from 30 to 72 weeks displaying egg production, elevated mortality rate, hyperimia in chicken abdomen, ovaries degeneration and enlargement of liver and spleen with fatty degeneration and amyloidosis.Up to now, there is no commercial vaccine for preventing avian hepatitis.In this study, we explored the express of a HEV ORF2 protein in lactococcus lactis, and evaluated the protective effect of the recombinant positive L. lactis expressing the ORF2 protein.The experimental methods and results were as follows:The ORF2 gene fragment coding the capsid protein of a HEV was cloned into prokaryotic expression vector p ET-30a(+) to construct plasmid p ET-30(a)-ORF2. The positive plasmids p ET-30(a)-ORF2 were transformed into the E. coli competent cells to screen positive strains. The screened strains were induced by IPTG, and then the expression of objective protein was detected by Western-blot. The purified protein was used to produce polyclonal antibody by immunizing New Zealand white rabbits. The gene fragment of antigen domain in ORF2 gene(△ORF2) was cloned into the vector p TX8048 to produce three plasmids p TX8048-△ORF2,p TX8048-SP-△ORF2-CWA and p TX8048-SP-△ORF2. The above plasmids were respectively electrotransformed into host strains L. lactis NZ9000 to select three positive bacteria L. lactis/p TX8048-△ORF2, L. lactis/p TX8048-SP-△ORF2-CWA and L. lactis/p TX8048-SP-△ORF2. The expression of the three objective proteins in positive strains were detected by Western-blot after the strains were induced by nisin. The results showed that the ORF2 protein was expressed in E.coli strain with molecular weight 72 k Da. The titer of the prepared polyclonal antibody was 216.The protein expression in positive strains L. lactis/p TX8048-△ORF2, L.lactis/p TX8048-SP-△ORF2-CWA and L. lactis/p TX8048-SP-△ORF2 could all be detected by Western-blot with the molecular weight 56 k Da for protein in bacteria, 67 k Da for protein on the surface of bacteria and 54 k Da for secreted protein.SPF chickens were immunized orally with 5 ×109 CFU recombinat positive bacteria induced by nisin including L. lactis/p TX8048-△ORF2, L. lactis/p TX8048-SP-△ORF2-CWA, L.lactis/p TX8048-SP-△ORF2 and L. lactis/p TX8048. The Ig G in serum and s Ig A in intestinal lavage fluid and bile specific for ORF2 protein were detected by indirect Enzyme Linked Immunosorbent Assay(ELISA) method. The m RNA level of cytokines IL-2 and IFN-γin spleen and liver of chickens immunized for three times were quantified by real-time PCR. The results showed that the level of Ig G in serum and s Ig A in intestinal lavage fluid and bile in chickens immunized by the three recombinant bacteria was significantly increased than that in chickens immunized by L. lactis/p TX8048 and in PBS control group(p < 0.01). The m RNA level of cytokines IL-2 and IFN-γ in spleen and liver of chickens immunized by the three recombinant bacteria were significantly higher than that in the group immunized by L.lactis/p TX8048 and in PBS control group(p<0.05 or p<0.01).After three immunizations, chickens in the four immunized groups were all challenged with a HEV by intravenous inoculation in wing vein. The absolute quantification of virus shedding in feces and virus load in serum of chickens in each group was quantified by real-time PCR, and the level of LDH in serum of chickens in each group was detected. The results showed that virus load in feces and serum of chickens in the groups immunized by the three kinds of recombinant bacteria expressing ORF2 protein was significantly higher than that in the groups immunized by L.lactis/p TX8048 and in PBS control group(p<0.05 or p<0.01). On day 7 post challenge, the LDH level in serum of chickens in the group immunized by L.lactis/p TX8048 was significantly higher than that in the groups immunized with the three kinds of bacteria expressing ORF2 protein(p<0.01).The chickens in each challenged group was systematicaly sacrificed, and the protection provided by immunization of three recombinant bacteria expressing ORF2 protein on pathological lesion caused by a HEV was evaluated based on pathology. The results showed that the target organs invaded by a HEV were liver and spleen, and the pathological lesion was aggravated with the increase of chickens age. On day 21 post challenge, both liver and spleen of chickens in the group immunized by L.lactis/p TX8048 were swell significantly, and the gross lesions in liver and spleen of chickens in the group immunized by the three recombinant bacteria were not significant.On day 28 post challenge, local congestion, petechial hemorrhage and lesion area with blister were observed in liver and spleen of chickens in the group immunized by L.lactis/p TX8048, and the gross lesions in liver and spleen of chickens in the group immunized by the other three recombinant bacteria were not serious as the group immunized with L.lactis/p TX8048. For the histopathological changes, the infiltration of inflammatory cells were observed in local area of liver, hepatocyte were vacuolar degeneration, necrosis and even cell disintegration. A large numberof red cell existed in white pulp of spleen, and cellulose exudation were also observed. The local necrotic foci with scattering distribution in white pulp were mainly the degenerated and necrotic lymphocyte, reticular cells and macrophage cells.The histopathological changes in liver and spleen of chickens in the group immunized by the other three recombinant bacteria expressing ORF2 protein were not significant.This study provide important references for the preparation of vaccine for avian hepatitis E,and for the further research of a HEV pathogenesis. |
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