Cell-Surface Display And Mucous Immunity Of The Protective Antigen Of The SLT-â…¡eB Of Pathogenic E.coli

Edema disease (ED) of weaning piglets is caused mainly by the pathogenic Escherichia coli which produce Shiga-like toxin type II variant (SLII-IIe). Presently, the gene coding for SLT-IIe has been cloned and sequenced, of which operon is 1986bp long and contains one A subunit (SLT-IIeA) and five B subunits (SLT-IIeB) . The A subunit is a toxic subunit with RNA N-glycosidase activity, leading to inactivation of the cellular protein synthesis and disorganization of metabolic functions of the cell. The B subunit contains the main protective antigen, of which specific antibody can prevent SLT-IIe from binding to its receptor.In this study, the author cloned the slt-IIeB and expressed the protein (SLT-IIeB) on the surface of E.coli HB101. Then, use the expressed protein as vaccine to excite the immune response on the surface of gut mucous.Both the coding sequence of SLT-IIeB (204bp) was amplified from PPSLT-IIeB named slt-IIeB and the coden sequence of the cell wall's fixed region of SPA (621bp, named SPA-X) was amplified from Staphylococcus aureus from animal. Then the two kinds of sequence (slt-IIeB and SPA-X) were linked and cloned into prokaryotic expression vector pEZZ-18 and resulting recombinants were called PZBSPAX.With the technology of immunofluorescence (IF) and Colloid Gold, the fusion protein was examined in different conditions such as tempreture and time. The result shows that the fusion protein(PP-SLT-IIeB-SPAX)were expressed on the surface of recombinant PZBSPAX. The SDS-PAGE and Western-blot also shows that the recombinant PZBSPAX have the expected expression.One-day-old piglets and 15-day-old piglets were separated by experimented group and control group. In experimented group, piglets were oral vaccinated by the recombinant PZBSPAX and the control group's piglets were oral vaccinated by nothing. The E.coli which resist ampicillin isolated from the experimented group's piglets on the lst 3rd, 7th, 15th and 30th day were identified by the specific recominant plasmid. The result shows that the recombinant PZBSPAX can live in the gut of experiment-group's piglets stably during the experiment. The gut-mucous specific SIgA and serum specific IgA and IgG were examined by ELISA on the 10th, 15th and 30th day. The result shows that the experiment-group's piglets can stimulate high level of mucous specific SIgA and serum specific IgA and IgG which indicates the recombinant PZBSPAX can act as oral vaccine to active the mucous immune respond and system humoral immune respond.This research has successfully construct the Gram-negative surface expression vector and through which the protective B subunit of SLT-IIe was expressed on the surface of HB101. The mucous vaccine can active mucous immune respond were seldom reported..So the author considers that the recombinant PZBSPAX can used as a potential mucous vaccine to protect the infection of swine edeman disease.