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.
Cao
Shouchun
S
National Institute for Control of Pharmaceutical and Biological Products, Beijing, 100050, China.
Zhang
Ying
Y
Liu
Feng
F
Wang
Qin
Q
Zhang
Quanfu
Q
Liu
Qinzhi
Q
Li
Chuan
C
Liang
Mifang
M
Li
Dexin
D
D000281:Administration, Intranasal; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D054307:Enterotoxigenic Escherichia coli / Q000235:genetics; D004768:Enterotoxins / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D005260:Female; D005677:G(M1) Ganglioside / Q000378:metabolism; D006476:Orthohantavirus / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014611:Vaccination / Q000379:methods*; D014765:Viral Vaccines / Q000276:immunology
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D014765:Viral Vaccines; D005677:G(M1) Ganglioside; C027538:heat-labile enterotoxin, E coli