Effects Of Salmonella Flagellin Subunit B To Different IBDV Immunogen Immunization

Salmonella flagellin, the structure of the thalli flagellum, is a monomeric form protein. It is not only the component of the movement and pathogenicity bacteria, but also the agonist of natural Toll-like receptor 5. The flagellin which can activate the MyD88 signaling pathway in combination with TLR5, can cause the increasing secretion of cytokines, such as IL-8, and can enhance the differentiation and proliferation and other effects of immune cell. Although the flagellin has Salmonella H epitope regions, effects to bring into full play of adjuvant function, and even can enhance the antigenicity as an self-adjuvant. Therefore, since 1998 the adjuvant effect of flagellin, has been a hotspot as vaccine immunization intensifier. Lactobacillus casei is the GRAS (generally regarded as safe) levels of probiotics. Because the thalline and secretion could significantly promote mucosal and humoral immunity, so it was a candidate as a living trager of vaccine carrier. The aim of the study is to explore immunologic enhancement of the pronucleus of recombinant flagellin, and explore immunologic enhancement of the different vaccine, antigen model.Overlap extension PCR method is applied to remove the hypervariable regions FliB (171 aa～ 407 aa), only keep consensus sequence of the amino terminal and ami no terminal, and insert the flexible peptide sequence and restriction enzyme cutting site, and we can obtain the FliBc, restructuring gene model of immunopotentiators, which is easy to plug in different antigen. Connectting the FliB and FliBc protein to the pPG-2 vector constructed the recombinant lacbobacillus casel pPG-2-FliB/L.casei 393 and the pPG-2-FliBc/L.casei 393, could inducted expression of secreting type recombinant protein, which was 53 kD and 55kD. Detecting the activity of TLR5 of Human colon epithelial cells Caco-2 in vitro showed that levels of expression of IL-8 increased significantly, and it showed that the recombinant FliB and FliBc protein had the bioactivity in vitro. And under the same culture conditions, the unit volume pPG-2-FliBc/L.casei 393 culture supernatant protein TLR5-stimulating activity was stronger than pPG-2-FliB/L.casei 393.First, in order to test the expression of a truncated protein FliBc after VP2 antigen on whether the enhanced immune effect, the IBDV VP2 gene was inserted protective antigen recombinant plasmid pProHTa-FliBc in E. coli expression system using the VP2-FliBc fusion protein, and with the other two forms of the protein (pure expression of VP2 protein, VP2 and FliBc recombinant protein expression, respectively, mechanical mixing) were added to Freund’s adjuvant subunit vaccine prepared model, testing the indicators SPF chickens immunized explore constructed FliBc is able to enhance the immune response induced by the antigen is inserted, and the effect of integration and mixing with the use of the antigen. Detection of immune chicken IgY serum titer chimeric protein VP2-FliBc group was significantly higher than VP2+FliBc mixed group and VP2 group, MTT assay spleen lymphocyte proliferation and attack virus protection rates have risen compared with the control group. Prove FliBc and VP2 antigen protein fusion after immunization has played a significant role in immune enhancement, but also further validate previous conclusions:FliBc antigen fusion protein antigen immune effect is stronger than the mechanical mixing, because FliBc in connection with the antigen can be better thereby enhancing the antigen presenting its specific immune response.In order to study the immunization effect of flagellin combined with attenuated vaccine, this experiment selected the commercialization of IBDV weak poison vaccine (D78 plant) as a model, with muscle injection (im) and intranasal (in) immune methods, respectively, and total the same recombinant protein content pPG-2/L.casei 393, pPG-2-FliB/L.casei 393 and pPG-2-FliBc/ L.casei 393 supernatant common immune 7-day-old SPF chickens immunized once. Weekly blood until 56 days after the detection of the immune serum IBDV specific IgY titers showed two ways pPG-2-FliBc/L.casei 393 supernatant groups have the most efficient price, and immunization in 49 days after SPF chickens with IBDV virulent strain (vvIBDV) were attack drug, calculate the rate was also significantly higher immune protection. pPG-2-FliB/L.casei 393 group also showed some immune-enhancing effect, but the effect is less than the truncated FliBc supernatant groups. Horizontal comparison of intramuscular and intranasal routes of immunization immune pathways, humoral and cellular immune responses were higher than the intensity of intramuscular way intranasal group, but the detection of tears and intestinal mucus secretory IgA (slgA) levels were significantly higher in group in im group, indicating flagellin in mucosal immunity have enough ways to enhance local immune function. After the detection of the immune serum neutralizing antibody titers, im set the overall level is higher than in, the highest neutralizing titer of 1:100; immune pathways within the same group pPG-2-FliBc/L.casei 393 was significantly higher than the control group group, while pPG-2-FliB/L.casei 393 group compared with the control group was not significant.FliBc evaluation of lactic acid bacteria as IBDV VP2 gene enhancer live vector vaccine:The VP2-FliBc gene pPG-2 connection transformed Lactobacillus casei L.casei 393, with Western-blotting method for detection of recombinant bacteria and culture supernatants were expressed consistent with the expected molecular weight of approximately 76 kD recombinant protein. The cultured cells were induced to 1010 CFU/mL, pPG-2-VP2-FliBc/L.casei 393 and this laboratory-built pPG-2-VP2/L.casei 393, and a control group pPG-2/L.casei 393 respectively SPF chickens immunized by the oral route, and strengthen the immune interval 10 d twice IgY specific serum antibody titers, pPG-2-VP2-FliBc/L.casei 393 group than pPG-2-VP2/L.casei 393 group; and after lymphocyte proliferation index and challenge protection rates were higher than pPG-2-VP2/L.casei 393 groups. Description IBDV VP2 gene insertion FliBc Lactobacillus casei live vector vaccine gene can enhance humoral and cellular immune responses of animals with immune-enhancing activity.After the detecting of the valency of specificity IgY, the results showed that levels of the recombination VP2-FliBc protein were significantly higher than that in VP2 and FliBc group. And the cell multiplication of splenic lymphocyte and the protection potency were higher than that in the control group. The results showed that recombination VP2 protein developed the immunologic enhancement obviously. Furthermore it showed that FliBc combined with antigen could well present the antigen to developed the immunologic enhancement.In summary, this study established the expression of Salmonella flagellin FliB full-length and truncated flagellin FliBc of E.coli expression system and Lactobacillus casei and correct expression of the two proteins, through the expression of two recombinant L.casei 393 protein in vitro activity compared display truncated FliBc of TLR5-stimulating activity stronger than FliB. While establishing three forms IBDV vaccine model:lactic acid bacteria live vector vaccine VP2 subunit vaccines, attenuated vaccines and commodities IBDV VP2 gene, respectively, will FliBc protein expression and gene fusion of form and three with the use of serum IgY, mucosal IgA and immune protection trials evaluating immune effect, indicating whether the three vaccine FliBc both play an enhanced role of humoral and cellular immunity, with the development of the immune adjuvant potential.