Study On The Enhancement Of Immunogenicity Of Prrsv-gp5 Protein By Ltb

【Objective】In order to study the adjuvant effect of the LTB, the expression conditions and purification methods of LTB and GP5 were optimized. On this basis, mice and piglets were immunized with model antigen-gp5 protein and using LTB as adjuvant.【Method】Prokaryotic expression of GP5 induction time, IPTG concentration, lysis buffer and the protein purification conditions were optimized. Eukaryotic expression of LTB induction time, methanol concentration, expression medium, initial concentration of bacteria and the protein purification conditions were optimized. The female ICR mice were divided into 8 groups of 10 each. The groups are PBS negative control group, GP5+ Freund's adjuvant positive control group, GP5 intramuscular group, GP5+LTB intramuscular group, GP5 subcutaneous group, GP5+LTB subcutaneous group, GP5 nasal drops group and GP5+LTB nasal drops group. The serum antibody was measured by ELISA and the lymphocyte proliferation was determined by MTT after the third immunization. The 1~2 days old piglets were divided into 4 groups of 10 each randomly. The groups are GP5 nasal drops group, GP5+LTB nasal drops group, GP5 intramuscular group and GP5 intramuscular+LTB nasal drops. The serum antibody was measured by ELISA after the second immunization.【Results】The optimal expression and purification conditions of GP5: induction time is 3.5h, IPTG concentra -tion is 1mM, lysis buffer is 8M Urea (pH7.8), wash buffer is 8M Urea (pH5.0) and 8M Urea (pH4.5), elution buffer is 8M Urea (pH4.0). The results of ELISA showed that the protein specifically reacted with PRRSV-positive sera from pigs, not negative sera. The optimal expression and purification conditions of LTB: induction time is 96h, methanol concentration is 1%, initial concentration of bacteria is OD600=4, expression medium is 1×BMMY, precipitation by 55% saturation of ammonium sulfate, the first affinity chromatography select Ni-NTA column, the second affinity chromatography select Immobilized D (+) -galactose column, the purity of eluted protein existed in pentamerand the results of ELISA showed that the protein specifically reacted with GM1.? The results of humoral immunological effect showed that LTB+GP5 protein group achieved higher levels of specific antibody than that of GP5 protein groups, but lower than that of Freund's adjuvant group. Intramuscular way proved a better immune effect than subcutaneous way and the immune effect of nasal drops group was not significant. The results of cellular immunological effect indicated that GP5+Freund's group, GP5+LTB intramuscular group and GP5+LTB subcutaneous group induced B lymphocyte proliferation significantly. The T lymphocyte proliferation was not observed in all groups except the Freund's adjuvant group. The results of piglets humoral immunological effect showed that LTB has a good mucosal immune enhancement, LTB+gp5 protein group achieved higher levels of specific antibody than that of GP5 protein groups, LTB nasal drops+GP5 intramuscular group proved a better immune effect than LTB+ GP5 nasal drops group.?【Conclusion】Prokaryotic expression and purification conditions of GP5 and Eukaryotic expression and purification conditions of LTB were confirmed. Mice Immunological tests showed that LTB has a good humoral immune enhancement, it can enhance the immunogenicity of GP5 protein by intramuscular and subcutaneous effectively. Piglet immunological tests showed that LTB has a good mucosal adjuvant, it can enhance the immunogenicity of GP5 protein by nasal drops too.