Study On The Safety Of Recombinant E. Coli Heat-Labile Enterotoxin B Subunit To Duck

Escherichia coli heat-labile enterotoxin B subunit(LTB) can combine with ganglioside GM1 receptors of epithelial cells. It possesses great immunogenicity and immune enhancement activity. LTB is regarded as one of the most popular mucosal immune adjuvant currently. Large scale production and safty performance are key to industriali-zation of LTB.To obtain effective expression of recombinant LTB, recombinant P.pastoris was cultured in automated fermentor with following conditions:culture temperature at 30℃, pH 6.0, dissolve of oxygen(DO) 35%, ammonia and 2% peptone as nitrogen source,1% methanol as carbon source. To maintain the methanol concentration at about 1.5%, methanol feeding rate was increased gradually according to the DO fluctuation curve after the initial carbon consumption. The result shows that expression of LTB peaked at 96 h post induction. The recombinant LTB was purified through ammonium sulfate precipitation and Ni2+ chelate affinity chromatography. The purity of LTB is up to 98%. Its pentameric structure was verified by denaturing and naturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Results of enzyme-linked immunosorbent assay (GM1-ELISA) indicated that recombinant LTB could bind GM1 ganglioside.To determine the dynamics of growth and decline of LTB, serum LTB content was measured at different time points after injection. The results showed that content changes and duration of LTB maintained a positive correlation with its injected dose. To evaluate the effect of LTB on immune function in ducks, immunoglobulins, cytokines and soluble CD4 and CD8 levels in serum were detected by ELIS A. High dosage of LTB could enhance IgG levels significantly (P<0.01), and increase IgA、IgM、IL-6 and IL-10 levels significantly (P<0.05) but with no significant effect on TNF-a (P>0.05). Serum soluble CD4 levels increased significantly (P<0.05) and CD8 levels decreased, but not significantly (P>0.05).Safety study included LTB protein-coding gene and antibiotic marker gene residue test, allergic reactions, immune response detection and study on the toxic effect of LTB. The results showed that no residues of LTB protein-coding gene and antibiotic marker gene were detected in purified LTB. LTB would not initiate allergic reactions in experimental ducks, but it could induce antibody response which is positively correlated to the administration dosage. Clinical symptoms and autopsy pathology were observed after three times intramuscular injection, and samples of heart, liver, spleen, lung, kidney and brain were collected for slice preparation, which were examined after coloration with haematoxylin and eosin(HE) or immunohistochemical(IHC) staining. Obvious cloudy swelling and degeneration of hepatocellular, severe inflammatory cell infiltration in lung and focal necrosis in brain tissue were observed in 100 mg/kg group. IHC staining results showed that LTB could be deteceted in all 6 kinds of examined organs and the strongest signal was observed in brain. This study defined the safty and the highest dosage of LTB for duck, also provided guidances for clinical application.