Objective To develop an efficient expression, purification system of recombinant Escherichia coli heat labile enterotoxin B subunit (rLTB) and study its activity against mucosal immunoadjuvant by nasal immunization Methods A recombinant, pMMB68 LTB was generated by cloning the LTB cDNA fragment into an expression vector (pMMB68) and transformed it into the host strain marine vibrio VSP60 The relevant target protein was identified using SDS polyacrylamide gel electrophoresis (SDS PAGE) and Western blot Sephacryl S 100 gel filtration chromatography was carried out for purification of rLTB in engineering bacteria VSP60 BALB/c mice received hen egg lysozyme (HEL) alone or combined with rLTB by nasal administration After three times immunization, IgG and IgA antibody levels in serum or small intestine wash samples were determined using ELISA Results rLTB protein was highly expressed in VSP60 After gel filtration with Sephacryl S 100, the purity of rLTB reached 98 1%, the yield rate was about 52% After immunization, IgG and IgA antibody responses specific to HEL in system and mucosa of HEL+rLTB groups were significantly increased, compared with the HEL alone group ( P <0 001) Conclusions A set of protocols for large scale rLTB preparation has been established, which is simple, efficient and applicable The rLTB protein we prepared was proved to be a powerful mucocal adjuvant, which could greatly enhance systemic and mucosal immune responses to nasally co administered antigen Objective To develop an efficient expression, purification system of recombinant Escherichia coli heat labile enterotoxin B subunit (rLTB) and study its activity against mucosal immunoadjuvant by nasal immunization Methods A recombinant, pMMB68 LTB was generated by cloning the LTB cDNA fragment into an expression vector pMMB68) and transformed it into the host strain marine vibrio VSP60 The relevant target protein was identified using SDS polyacrylamide gel electrophoresis (SDS PAGE) and Western blot Sephacryl S 100 gel filtration chromatography was carried out for purification of rLTB in engineering bacteria VSP60 BALB / c mice received hen egg lysozyme (HEL) alone or combined with rLTB by nasal administration after three times immunization, IgG and IgA antibody levels in serum or small intestine wash samples were determined using ELISA Results rLTB protein was highly expressed in VSP60 After gel filtration with Sephacryl S 100, the purity of rLTB reached 98 1%, t He yield rate was about 52% After immunization, IgG and IgA antibody responses specific to HEL in system and mucosa of HEL + rLTB groups were significantly increased, compared with the HEL alone group (P <001 1) Conclusions A set of protocols for large scale rLTB preparation has been established, which is simple, efficient and applicable The rLTB protein we prepared was proved to be a powerful mucocal adjuvant, which could greatly enhance systemic and mucosal immune responses to nasally co administered antigen