The Construction Of Recombinant Lactic Acid Bacteria Expressing ΔHexon Protein And Its Immune Protective Effects Against FadV-4 Infection

Hepatitis-hydropericardium syndrome（HHS）is an infectious disease caused by Fowl adenovirus 4（FAd V-4）infection,which caused heavy economic losses to the global poultry industry.Since 2015,the disease has broken out one after another in various parts of China.Currently no commercial vaccine is available in China,and the control means of the disease in the world mainly rely on the inactivated vaccine and attenuated live vaccine.However,due to the inactivated vaccine and attenuated live vaccine’s potential risks,subunit vaccine research as a new type of vaccine has gradually become a hot spot.Lactic acid bacteria a kind of food-grade probiotics,showed significant advantages in human and animal vaccine development and become a suitable carrier for vaccine research.Based on previous studies,we used two different bacteria,including Lactococcus lactis NZ9000（commercial lactic acid bacteria）and E.faecalis MDXEF-1（Enterococcus faecalis isolated by our laboratory）to express the main antigenic determinants of FAd V4ΔHexon protein.The antigenicity was expected to be enhanced,involving the increased target antigen’s uptake by dendritic cells and the more robust specific immune responses through fusing dendritic cell-targeting peptides with bacterial surface anchoring.In this study,the effect of recombinant lactic acid bacteria against FAd V-4 was evaluated by inoculating 7-day-old chickens via oral route to test the level of immune response and immune protection against after challenge.In this study,the target geneΔHexon was cloned from virus genomic DNA,and the recombinant E.coli bacteria BL21/p ET30a-ΔHexon was constructed by chemical transformation.At the same time,the recombinant lactic acid bacteria were constructed by electrotransformation.The recombinant bacteria BL21/p ET30a-ΔHexon was further induced and followed by SDS-PAGE seperation、protein purification and renaturation.Then polyclonal antibodies againstΔHexon protein were prepared by immunizing New Zealand rabbits with the renaturedΔHexon protein,and the antibody titers were detected by ELISA.The antibody specificity was verified by reacting with liver protein of FAd V-4-infected chickens using Western blot.The expression of target protein in lactic acid bacteria was detected by Western blot method,and the surface expressedΔHexon protein was verified by bacterial indirect immunofluorescence.The results showed that theΔHexon gene fragment of 1230 bp was cloned,and 51 k Da protein was expressed in BL21.The titer of rabbit polyclonal antibody reached 2¹⁶ and the target band of 110 k Da could be detected in liver protein of FAd V-4-infected chickens by Western blot.The results of Western blot and bacterial immunofluorescence experiments showed that the recombinant bacteria displayed the target protein in the form of surface anchoring.Secondly,after induction by Nisin,the colony concentration of recombinant lactic acid bacteria was adjusted to 1.0×10^{1 1} CFU/m L for L.lactis NZ9000 and 5.0×10^{1 0} CFU/m L for E.faecalis MDXEF-1.Chickens in the control group were immunized with the same dose of PBS（p H 7.2）.The lavage fluid in jejunum and serum from chickens in each group were prepared at 7,21,35,and 49 days after oral administration.The Ig G levels in serum and s Ig A levels in lavage fluid of jejunum were detected by indirect ELISA.The peripheral blood of each group was sampled to prepare lymphocyte suspension on 14 day after tertiary immunization（49 days old）,and the lymphocyte proliferation was detected by CCK8 kit.The spleen of each group was sampled on 14 day after the third immunization,and the m RNA expression levels of IL-2、IL-4、IL-10、and IFN-γwas detected by real-time quantitative PCR（q RT-PCR）.The results showed that the immunization of the four recombinant bacteria could induce chickens to produce antigen-specific Ig G and s Ig A.The antibody levels stimulated by the recombinant bacteria harboring DCpep was significantly higher than that of the recombinant bacteria without DCpep.After three immunizations,the proliferation of peripheral blood lymphocytes and the m RNA relative expression of IL-2、IL-4、IL-10 and IFN-γin the spleen of chickens immunized with the four recombinant bacteria was significantly higher than that in the control group and PBS group.The above indexes in the group immunized with recombinant bacteria harboring DCpep increased significantly.Finally,in order to evaluate its immune protective effects,at two weeks after the third immunization（49 days old）all chickens except in PBS group was subcutaneously infected with FAd V-4/GX01 10^5.2ELD₅₀.And the changes of enzymes related to liver function、virus load in liver、pathological changes in liver、spleen、heart、kidney and liver function were detected after challenge.Taken together,displaying the target proteinΔHexon and fusion protein of dendritic cell tar-geted peptide andΔHexon on the cell surface of L.lactis NZ9000 and Enterococcus faecalis MDXEF-1 can enhance the immunity of the body,which is a potential candidate against FAd V-4infection.