Expression Of The Mutant Escherichia Coli Heat-labile Enterotoxin And Study Of Its Effect On Mucosal Immunity

Heat-labile enterotoixin(LT),produced by Enterotoxigenic Eshchirichia coli(ETEC),is the virulence factor of diarrhea in humans and animals.More interesting,LT also possesses strong immunogenicity and can assist other antigens delivered through mucosal pathway to produce specific antibodies,so it is a good mucosal immunization adjuvant and has significant medical and economic values in mucosal immunization research and development of vaccine for mucosal immunization.In order to obtain non-toxic mutant of LT retaining the mucosa immunological adjuvant activity, three pairs of primers were designed based on the published nucleotide sequence of LT.The first and second pairs of primers were ueed to genetically modify LT-A subunit by mutation at residues63(Ser-Lys)using the overlap extension PCR.The third pairs of primers were used to amplify sequence of LT-B subunit. The PCR products was cloned into pMD18-T Simple Vector respectively. The recombinant plasmids were identified to be containning the mutant gene by the methods of restriction endonuclesae digestion and sequencing. Then the mutant LT-A gene and LT-B gene were cloned into prokaryotic expression vector pET-30a(+).The recombinant plasmid was transformed into E.coli BL21and about50KD of target protein was expressed with expectation.This suggests that recombinant E.coli BL21-pET-LTS63K was successfully built and can be used for the study of its adjuvant activity.Based on related studies,the extracellular domain of CTLA-4s remains the binding ability to B7molecules without providing negative signal and thus can target the fused antigens to antigen presenting cells.we fused LTS63K and CTLA-4IgV together to study the adjuvant activity of the fusion protein. LTS63K sequence built in the former research was cloned into expression vector pET-CTLA-4IgV and the recombinant expression vector pET-CTLA-LTS63K was transformed into E.coli BL21. After induction with IPTG,the recombinant E.coli expressed an expected60KD fusion protein CTLA-LTS63K based on SDS-PAGE analysis. Western blotting showed that the expressed proteins of LTS63K and CTLA-LTS63K were recognized by anti-LT serum.This proved the expressed proteins had good reaction activity.The fusion proteins were present in the pellet fraction of the bacterial lysate and purified as inclusion bodies by repeated washing in water and PBS containing2M urea.The soluble forms of the two fusion proteins were obtained by denaturation/renaturation in8M urea. In order to study the adjuvant activity to Inactivated vaccine of the two proteins,Avian influenza inactivated vaccine and protein LTS63K or CTLA-LTS63K were immunized with the co-administrion of intramuscular injection.180healthy7-day-old chickens were assigned to6groups. The first group and the second group were intramuscularly immunized by the AIV inactivated vaccine with different dose of LT mutant as adjuvants. The third group and the fourth group were intramuscularly immunized by the AIV inactivated vaccine with different dose of CTLA-LT fusion protein. The fifth group was only immunized by the inactivated vaccine. And nothing has been done on the sixth group as control. The antibody specific for AIV was determined every7days after the immunization. It was showed that the two proteins could not significantly improve the serum antibody titer and mucosa antibody titer.To study the mucosal adjuvanticity of the mutant protein LTS63K and the fusion protein CTLA-LTS63K on immune response,240healthy7-day-old chickens were assigned to8groups. The1st,2nd and the3rd group were orally immunized by the IBDV attenuated vaccine with different dose of LT mutant as adjuvants. The4th,5th,6th group were orally immunized by the IBDV attenuated vaccine with different dose of CTLA-LT fusion protein. The7th group was immunized by the attenuated vaccine with normal saline water. And nothing has been done on the8th group as control. The serum and mucosal antibody specific for IBDV was determined every7days after the immunization. It was showed that both the two proteins could significantly improve the serum antibody titer and mucosa antibody titer.