Construction Of Recombinant Lactic Acid Bacteria Expressing Variant Strain Of Chicken Infectious Bursal Disease Virus VP2 And Evaluation Of Its Immune Effect

Lactic acid bacteria usually refer to a type of gram-positive bacteria that can metabolize sugars into organic acids such as lactic acid that exists in nature and in the mucous membranes of humans and animals.The biological and food safety of some lactic acid bacteria is recognized worldwide.The probiotic lactic acid bacteria do not contain endotoxin,its cell wall and nucleic acid and other components have immune adjuvant effect,so lactic acid bacteria are the first choice for expressing foreign antigens.Infectious bursal disease（IBDV）is a disease that seriously harms poultry and causes immunosuppression.Recently,a variant strain of the infectious bursal disease virus（IBDV）has been identified in China,Japan and North America.The IBDV vaccine cannot protect against infection by the variant IBDV（vaIBDV）.Therefore,this study intends to conduct research on new vaccine by constructing recombinant lactic acid bacteria expressing variant IBDV VP2 as a candidate for preventing vaIBDV.In this study,the biosafety and food safety grade probiotic Lactococcus lactis NZ3900 strain was used as the host bacteria,and the antibiotic-free food safety grade p NZ8149 expression plasmid vector was used to generate a recombinant Lactococcus lactis expressing vaIBDV VP2（vaVP2）.In order to improve the uptake of recombinant vaVP2 protein by antigen-presenting cells,we fused vaVP2 with part of the functional domain of Salmonella typhimurium invasion protein RCK,and constructed recombinant Lactococcus lactis.Western blot showed that recombinant Lactococcus lactis could successfully express the protein with a size of about 52 k D;protein quantitative detection showed that the recombinant protein was 57.6μg/m L.The results of animal experiments showed that each chick was injected with 10⁸ CFU of recombinant Lactococcus lactis-vaVP2-RCK.Two weeks after immunization,the serum neutralizing antibody titer against vaIBDV-SHG19 was 1:2^4-6,while the antibody titer against vv IBDV-HLJ0504was 1:2^1-2;the challenge results showed that the SPF chickens vaccinated with recombinant Lactococcus lactis-vaVP2-RCK had 100%immune protection against vaIBDV-SHG19,the BBIX index was greater than 0.7,and the bursa of fabric was not affected.the SPF chickens vaccinated with recombinant Lactococcus lactis-vaVP2-RCK could also provide 100%survival against vv IBDV-HLJ0504,but the bursa of fabric was damaged to a certain extent,and the BBIX index was almost all less than 0.7.Detection of the proliferation activity of T lymphocytes and antigen-presenting cells in immunized chicken peripheral monocytes（PBMCs）showed that immune recombinant Lactococcus lactis-vaVP2-RCK can significantly increase the number of chicken peripheral monocytes after two weeks.The proliferation activity of T lymphocytes and antigen-presenting cells;and cytokines IL-12 and interferon-γ（IFN-γ）in the chicken serum were significantly increased after post-three weeks of immunization and one week of attack.The above results indicated that the immune recombinant L.lactis r-L.lactis-vaVP2-RCK could play a complete immune protection against vaIBDV,and it could also play a certain immune protection against vv IBDV-HLJ0504.The recombinant Lactococcus lactis-vaVP2-RCK constructed in this study could induce unique neutralizing antibodies produced by the injection immunization,which could completely resist the infection of the mutant IBDV,and has important practical significance for the prevention and control of vaIBD.