Construction And Mucosal Adjuvant Activity Of Escherichia Coli Heat-labile Enterotoxin Double-mutants

Escherichia coli heat-labile enterotoxin (LT) generated from enterotoxigenic Escherichia coli (ETEC) is known to be a potent mucosal immunogen and immunoadjuvant towards co-administered antigens, but LT is toxic. toxicity must be declined or removed in order to utilize the adjuvant activity,. The key sites related to toxicity 63, 72, 192 were selected for double-mutants. The bioactivities of the double-mutants were tested and layed foundation for further adjuvant.LTK63/G192 and LTR72/G192 were made by gene SOEing PCR using pET30aLTK63,pET30a-LTR72 as template. Destination genes and pET30a were excised by NcoI and SalI and then inserted into pET30a. Recombinant plasmids were transformed into E. coli DH5αrespectively. The positive recombinant plasmids LTK63/G192 and LTR72/G192 were selected by PCR, restriction analysis and nucleotide sequencing which indicated the replacement AGA (192th) with GGA. The positive plasmids were transformed into the host E. coli BL21(DE3). E.coli BL21 (DE3) was induced to express by IPTG.The expressed proteins were identified by SDS- PAGE and Western-blot using Anti-His tag antibody.protein subunits LTA and LTB reacted with Anti-His tag antibody in Western-blot detection, The results showed that the two protein subunits were expressed successfully and the expressed protiens have the same immune activity as wtLT protein. The proteins were purified by Ni-NTA resin. The ADP-ribosyltransferase activity of wtLT and mutants treated with trypsin was determined using p-diethylamino-(benzylidinea-mino)-guanidine (DEABAG) as substrate. ADP-ribosyltransferase activity of the mutants was less than wtLT and approaches in the negative control group, LTK63/G192, LTR72/G192 double-mutant enzyme activity is also close. The toxicity assay of Patent-mouse showed that LTK63/G192,LTR72/G192 toxicity were all significantly reduced, LTR72/G192 toxicity is higher than LTK63/G192. LTK63/G192, LTR72/G192 and LT compare the difference to be the most significant. LTK63/G192, LTR72/G192 and PBS are more significant.than the difference.The ability of LTK63/G192,LTR72/G192,LT to act as a mucosal adjuvant was tested by immunizing mouse and chickens intranasally (i.n.), using Newcastle vaccine for chickens as a bystander antigen.That results showed that mucosal adjuvanticity of LTR72/G192 were most significant. Proliferation of spleen T cells of chickens induced by NDV, test showed: LTR72/ G192 + NDV is the most effective in proliferation react.Thus double-mutants of heat-labile enterotoxin can act as effective intranasal mucosal adjuvants.