The Effects Of Heat-Labile Enterotoxin B Subunit Of Escherichia Coli (LTB) On Oral Vaccination Of S.Iniae In Tilapia

Oral vaccination is a simple, efficient way of inducing immunity at mucosal surfaces. The slow development of oral vaccines has been mainly due to the lack of suitable delivery systems and an effecttive absorptive system. The heat-labile enterotoxin B subunit of Escherichia coli (LTB) is a potent signalling molecule and carrier which capable of modulating immune responses and usually used as an adjuvant in VP2, HEL, HP and et al.To investigate the enhancing of LTB which had been successfully constructed and expressed by KM71pichia pastoris strain, the recombinant proteins of LTB were induced express.In the present study, The LTB’s induced expression time was optimized and the toxicity was tested. The effects of LTB on oral vaccine of S. iniae in tilapia was investigated by an indirect enzyme-linked immuno-sorbent assay model which was newly established. The results showed that an enhancement can be facilitated when the vaccine is linked to LTB, LTB is an efficient adjuvant to S. iniae oral vaccine mucosal.In this study, The LTB’s induced expression time and the toxicity were observed.Studies indicated that LTB is low toxicity and well-induced express in the96th hour with the induced expression of2.716mg/mL. In the newly established mode of ELISA, the best optimum reaction temperature, concentration of antigen, serum dilution, multiple resistant dilution and Goat anti-rabbit IgG-HRP dilution are22℃,5.5μg/mL,1/400,1/2000and1/8000.Then the enhancement of LTB was evaluated in tilapia through the methods of oral vaccine with S. iniae vaccine and intraperitoneal injection (IP). The tilapia were designed vaccine with0.2×109cells/fih,0.2×108cells/fish and 0.2×107cells/fish three S. iniae vaccine doses combine with different LTB doses in1.68μg/fish,0.84μg/fish and0.17μg/fish. The safety and ELISA studies revealed that the best optimum combination S. iniae vaccine and LTB doses were0.2×108cells/fish and0.84μg/fish. When IP vaccinated fish were challenged by107strains of S. iniae, relative percent survival (RPS) values of vaccinated fish at10,20and30days were65.00,68.42and64.71%respectively. Mucosal antibody levels basically proportional to the LTB dose but range at a high fluctuation. LTB could effectively improve the lysozym and AKP, but inhibite SOD.In conclusion, the adjuvant of LTB plays a role in stimulating the effect of S.iniae oral vaccine of tilapia.Therefore LTB have the characteristic of a adjuvant to the S. iniae oral vaccine.