Construction Of Invasive Lactic Acid Bacteria Expressing ALV-J Gp85 Protein And Evaluation Of Immune Protection Effect

Avian Leukosis（AL）caused by Avian Leukosis Virus（ALV）is a kind of infectious tumor disease.At present,ALV can be classified into A-K 11 subgroups,of which J subgroup Avian Leukemia Virus（ALV-J）is the most infectious and pathogenic.J subgroup Avian leukemia caused by ALV-J as a worldwide distribution,mainly presents as tumor diseases in vivo and vitro,reproductive disorders and immunosuppression,which seriously causes immeasurable losses to the global poultry industry.So far,there is no an effect and commercialized vaccine for preventing and controlling the disease,and it is difficult to rely on the purification of breed flock to eliminate ALV-J.Therefore,it is imperative to develop a new and effective vaccine against ALV-J.Lactic Acid Bacteria（LAB）as a probiotic can colonize in the intestinal,and has probiotic effects such as maintaining the balance of intestinal flora and regulating immunity,so it is an ideal vector for delivering vaccine.Studies have shown that the invasive LAB expressing Staphylococcus aureus fibronectin binding protein A（FnBPA）could effectively enhance the immune response in mice and chickens during the delivery of the antigens of influenza virus and coccidium,and produce effective protective effects.Gp85 protein as the envelope protein on the surface of ALV,contains an antigenic determinant that binds to the receptor of cell surface in host.In this study,an invasive LAB expressing Gp85 protein of ALV-J was constructed,namely,NC8-pSIP409-FnBPA-pgs A’-gp85,and the immunoprotection efficiency of the recombinant strain against ALV-J was evaluated after orally immune with chickens.Detailed research contents and results was as follows:The gp85 gene was connected to the vector pSIP409-pgs A’to construct the recombinant plasmid pSIP409-pgs A’-gp85 by Seamless Cloning Kit.Taking the plasmid as template,the pgs A’-gp85 gene was amplified,and then was connected to the vector pSIP409-FnBPA to construct the recombinant plasmid pSIP409-FnBPA-pgs A’-gp85.And the plasmid was electrotransformated into NC8 to obtain the recombinant strain NC8-pSIP409-FnBPA-pgs A’-gp85,and the cell proteins were obtained by repeated freezing and thaw method.The expression of Gp85 protein was verified by Western-blot,and the results showed that the Gp85 protein was successfully expressed on the surface of NC8,and the size of the protein was about54.56KDa.In order to further evaluate the adhesion and invasion ability of the recombinant bacteria to DF-1 cells,the recombinant bacteria were co-cultured with DF-1 cells.The results showed that the adhesion rate of NC8-pSIP409-FnBPA-pgs A’-gp85（FnBPA-gp85）to DF-1 cells was 1.8%,and the invasion rate was 1.0%,which was significantly higher than that of NC8-pSIP409（409）（P<0.01,P<0.001）,but lower than that of NC8-pSIP409-FnBPA（FnBPA）（P<0.01,P<0.05）.FnBPA protein due to the bigger molecular weight,might affect the biological characteristics of recombinant strains.Thus,the strains,FnBPA-gp85 and FnBPA and409 were respectively subjected to the condition of the p H value of 2.5 and bile salts mass fraction of 0.5%of MRS medium.The results showed that the survival rate in FnBPA-gp85 group was more than 110%in acid environment,and more than 170%in bile salt solution.But there was no significant difference compared with 409（P>0.05）.Then,the plasmid stability of the recombinant strain was detected.The plasmid stability was close to 100%by spread plate method,and the gp85 gene was detected by PCR every 10 generations.Finally,the growth performance of the recombinant strain was tested.The results showed that the growth performance of the strain FnBPA-gp85was excellent.These results showed that the expression of FnBPA protein had little effect on the resistance of acid and bile salt and the stability of plasmid and growth performance.The experimental animals were divided into three groups:the PBS group,the FnBPA group and the FnBPA-gp85 group.Chickens at the age of 1-3 days and 14-16days were orally immunized with LAB at a dose of 2×10⁹CFU/200μL.At the age of30,except for the Control group（the part from the PBS group）without challenge,every chicken in other three groups were injected intramuscularly with ALV-J HB_2010001at a dose of 7×10^5.6TCID₅₀/700μL,Before challenge,the expression of the CD3⁺CD4⁺and CD3⁺CD8⁺T lymphocytes in the peripheral blood and spleen were detected by flow cytometry.T lymphocyte proliferation in spleen was determined by MTT,and the content of antibody Ig G cytokines,α-interferon（IFN-α）,IL-2,γ-interferon（IFN-γ）in serum and intestinal mucosal antibody s Ig A were detected by enzyme-linked immunosorbent assay（ELISA）.After challenge,the body weights of the chickens were recorded weekly,and the infection and cloacal swabs of chickens were tested every week.The organ indexes of chickens were measured at the 7th week after challenge.Finally,the relative virus loads of the organs of each group were compared.The protective effect of the recombinant strain against ALV-J was comprehensively evaluated.The results of animal experiments showed that the percentages of CD3⁺CD4⁺T lymphocytes of peripheral blood in groups FnBPA-gp85 and FnBPA were significantly higher than that of PBS group（P<0.001,P<0.001）,and there was no significant difference between the groups FnBPA-gp85 and FnBPA（P>0.05）.The percentage of CD3⁺CD8⁺T lymphocytes in peripheral blood of FnBPA-gp85 group was significantly higher than that of FnBPA group and PBS group（P<0.001,P<0.001）.The expression of CD3⁺CD4⁺T lymphocyte of spleen in FnBPA-gp85 group and FnBPA group was significantly higher than that in PBS group（P<0.001,P<0.001）,and the expression of CD3⁺CD8⁺T lymphocytes in FnBPA-gp85 group was higher than that in group FnBPA and PBS group（P<0.01,P<0.001）.The lymphocytes proliferation of spleen in FnBPA-gp85 group was significantly promoted after being stimulated by ALV-J Gp85protein.The ELISA results showed that the content of Ig G in FnBPA-gp85 group was significantly higher than that in groups FnBPA and PBS（P<0.001,P<0.001）.The content of mucosal antibody s Ig A in FnBPA group was higher than that in PBS group（P<0.01）,and significantly higher than that in PBS group（P<0.001）.The levels of cytokines of IL-2 and IFN-γin FnBPA-gp85 group were significantly higher than in groups 409 and FnBPA（P<0.01,P<0.01）.These results indicated that the recombinant LAB could enhanced the cell mediated immunity and humoral immunity and mucosal immunity and increase the expression levels of cytokines of IL-2 and IFN-γ.After challenge,the net weight gain of FnBPA-gp85 group was higher than that of groups FnBPA and PBS.The ratios of infection and cloacal swabs in FnBPA-gp85group were the lowest,and there was no significant difference in organ indexes between FnBPA-gp85 and Control group（P>0.05）.The relative virus loads of organ in FnBPA-gp85 group was lower than the groups PBS and FnBPA.And there were differences in the virus loads of the organs,heart and liver and kidney and bursa between the groups FnBPA-gp85 and PBS（P<0.05,P<0.01,P<0.001,P<0.001）.The relative virus loads of liver,spleen and bursa in FnBPA group were lower than the PBS group（P<0.05,P<0.01,P<0.001）,except for the spleen slightly lower than FnBPA-gp85 group,the virus loads of other organs were higher than in FnBPA-gp85group.These results indicated that the strain FnBPA-gp85 could to some extent produce the protective effect against ALV-J.The invasive Lactobacillus plantarum expressing the Gp85 protein of ALV-J in the study could effectively enhance the cell mediated immunity,humoral immunity and mucosal immune response,and promoted the levels of cytokines of IL-2 and IFN-γ,improved the capacity for resisting ALV-J in chickens,which laid the foundation for later exploring protection mechanism of LAB live vaccine resisting ALV-J.