Recombinant Escherichia Coli Heat-labile Enterotoxin:Preparation And Adjuvant Activity On Murine

LT is a kind of enterotoxin produced by some strains of Escherichia coli and is known to have powerful mucosal adjuvant activity. But it is unsuitable for clinical use because LT is very potent enterotoxins in it’s native state. Site-directe mutagenesis is used in exploring point mutations of LT aiming to reduce or eliminate the enterotoxicity of LT while maintaining it’s adjuvanticity. Former study in our lab has conducted plasmid pET30a-LTRG to produce recombinant LT, but it was expressed in inclusion bodies in E. Coli. As the recovering protocols is complicated and much of the peotein was lost in the process, we tried to do some research by expressing recombinant LT in soluble form. We also tried to improve the refolding process of LT expressed in inclsion bodies,The gene of the recombinant LT was amplified by PCR from the plasmid pET30a-LTRG (R72G192) and subcloned into the cold shock expressing vector pcoldⅡ to construct plasmid pcoldⅡ-LTRG. The recombinant plasmid was then transformed into the compound cell BL21(DE3) and induced. The expression of the recombinant protein was identified by SDS-PAGE and Western Blot, and the results shows that the expressed protein accounted for about10%of the total cellular proteins.Then the the recombinant protein was expressed in big scale and purified by nickel affinity chromatography.To improve the protein yield, the recovering of the protein expressed in inclusion bodies was conducted under different pH and denaturants using two methods: hyperfiltration and dilution. The results shows that the yields of recombinant protein under pH8.0、pH8.5、pH9.0was18.2%、28.8%、40.2%respectively when using the method of hyperfiltration, the yield was33%when using the method of dilution with Glycin-HCl buffer.Then the purified LTRG was coadministered to groups of mouse with NDV vaccine and the IgG in serum, IgA in Nasal Washes and HI antibody were examined by Elisa. The results shows that the soluble LTRG and protein recovered from inclusion bodies under pH value of9.0shows no adjuvanticity, the protein recovered under pH of8.0showed the most potent to induced serum anti-NDV IgG responses in immunized mouse and the protein recovered under pH of8.5showed the most potent to induced mucosal IgA responses. And the adjuvanticity of the protein recovered by dilution method were significantly hither than blank goup and the hyperfiltration groups.The in vivo toxicity of recombinant LT was evaluated by histology methods after nasal vaccination. The histological study shows that no pathological change in nasal mucosa, trachea, lung, liver or kidney was find with the groups of mouse immunized with recombinant LT, but the mouse received wild type LT develops inflammal pathological changes in lung such as lesions of Alveolar wall thickening, and inflammatory cell infiltration and bleeding. So the toxicity of the recombinant LTRG is significantly small than wild tipe LT.In conclusion, the collection of study expressed LTRG in soluble form and established a effective reconstitution system for recombinant LT from inclusion bodies, the obtained LT is shown to augment the IgG and IgA responses when delivered via intranasal route. the toxicity of the recombinant LTRG is significantly small than wild tipe LT delivered intranasally in histology research. It laid the base for follow-up exploitation of recombinant LTRG as mucosal adjuvant.