Cloning, Expression, Purification And Immunity Activation Of Escherichia Coli Heat-labile Enterotoxin B Subuint

The most powerful mucosal immunogens recognized to date are cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT), the molecules that cause the debilitating watery secretions typical of cholera and traveller's diarrhoea, respectively . The anti-toxin response induced is so potent that a strong immune response is also activated against foreign bystander molecules that are present simultaneously at the mucosal surface. Nevertheless, the use of LT as mucosal immunogens and adjuvants in humans has been limited by their toxicity. The three-dimensional structure of LT has been solved and these results have prompted attempts to dissect, using genetic techniques, the mucosal immunogenicity and adjuvant activity of LT from their toxicity. Such studies have allowed to design non toxic but immunogenic molecules and to define the roles of the receptor-binding B domain, the A subunit, and the enzymatic activity of LT in mucosal immunogenicity and adjuvanticity.To study the immunoadjuvanticity of recombinant Escherichia coli heat -labile enterotoxin B subunit (rLTB), we expressed LTB in E.coli and tested the antibody that generated after immune, by influent vaccine or HSV-II together with LTB.Firstly, we amplified the LTB·gene from large plasmid that extracted from ETEC 44815# strain by PCR. Then, we cloned this original PCR product into pMD18-T vector for sequencing, whose result showed that the gene wecloned was exactly LTB. Secondly, we tried to use pET21b(+) vector to express LTB in Escherichia coli BL2KDE3). Positive colony was confirmed by PCR screen and expression test. LTB protein was successfully expressed and its molecular weight on the gel of SDS-PAGE is 14KD which is consistent with the theoretical value.To test the immunoadjuvanticity of LTB, we performed large scale purification of this protein and used it as immunoadjuvant to immune mice together with influenza virus vaccine and HSV-II (gD) antigen respectively, which was controlled by without LTB and with Al(0H)3 as the adjuvant. BalB/C mice were administered via inject o or nasal rout. Collection of mucosal fluids, Nasal, lung, and gut washes were collected 3 days after the immunization. Nasal, lung, and gut lavages were performed on the sacrificed animal by repeated flushing by phosphate buffered saiine (PBS) containing 0.1% bovine serum albumin (BSA). Serum samples and vaginal fluids were collected 3 days after the last immunization.We use ELISA to test the antibody that generated after the immunity and killed the animal to test slgA from nose, lung, intestines and vagina or IgG, IgA, IgM from serum. The results demonstrated that LTB is an effective mucosal immunoadjuvant and its adjuvant activity is stronger than the control.