Secretory expression and purification of recombinant Escherichia coli heat-labile enterotoxin B subunit and its applications on intranasal vaccination of hantavirus.

In order to further study the B subunit of the Escherichia coli heat-labile enterotoxin (LTB), we obtained the LTB gene from pathogenic E. coli, cloned it into the pET22b (+) prokaryotic expression vector, and expressed it as a fusion protein with His tag in E. coli BL21 (DE3). The recombinant LTB was expressed and purified by Ni2+ affinity chromatography. The biological activity of the purified recombinant LTB was assayed in a series of monosialotetrahexosylganglioside (GM1)-ELISA experiments. The recombinant LTB (rLTB) was efficiently expressed under the induction of 10 g/l lactose at 37 degrees C for 6 h and yielded up to 31% of the total bacterial protein. Fused with pelB signal peptide, rLTB was successfully localized to the periplasmic space. GM1-ELISA experiments showed that the rLTB obtained retains strong GM1 ganglioside-binding activity. The ELISA result of hantavirus nucleoprotein-specific secretory immunoglobulin A (sIgA) and IgG showed that intranasal administration of inactivated hantavirus with rLTB significantly increased the levels of hantavirus-specific sIgA (P<0.01) and IgG (P<0.01) in comparison with inactivated hatavirus alone. In summary, we have developed a method for the efficient secretory expression and purification of rLTB, and the inactivated hantavirus co-administered intranasally with rLTB could effectively induce both mucosal and humoral immune responses specific to hantavirus.