Research On An Oral Live Vaccine Expression EV71 Antigen Gene VP1

Enterovirus type 71(EV71) is one of the major etiological agents of hand-foot-mouth disease (HFMD), which often causes severe neurological corqplications. Vp1 is the most important antigenic genes of EV71, it's product as a king of outer capsid protein takes important role in the infection of the host cell. So we choose vp1 as the target gene for an oral vaccine. LTB, a non-toxic subunit, is reported has powerful immunogen and adjuvant, so we expressed it with VP1 as intramolecular adjuvant in this study.Bifidobacterium exist in human intestinal tract normaly. It is an important beneficial bacterium, which has an important regulating function to the microenvironment of human intestinal tract and a special protection to the intestinal tract of an infant. Bifidobacteria can adhere to the gut. So we try to develop it as a gastrointestinal tract administers live vaccine system carrying EV71 VP1 and LTB of ETEC.ObjectiveIn order to develop HFMD vaccine preventing EV71 infection, the recombined expression vector for the vp1-ltb fusion gene was constructed and expressed both in E.coli BL21 (DE3) and Bifidobacterium. The immunicity of the recombinant Bifidobacterium was analyzed with ELISA. MethodsEV71 Vp1 gene was synthesised with 14 pairs of primers and the ltb gene fragment of ETEC (44815) plasmid DNA was amplified by PCR. Two fragments were linked by overlap PCR and the product was identified by sequencing analysis and inserted into the expression vector pBEX. The constructed recombinant plasmid pBEX-VP1-LTB was transformed into E.coli BL21(DE3). The expression product of VP1-LTB was analyzed by SDS-PAGE and Western blotting. The mice were immunized with the expressed product. The IgG and IgA levels were determined in sera, faces and intestines mucus to identify the immunogenicity of the expressed product. Then the recombined plasmid was transformed into Bifidobacterium by electroporation. The antigenicity of target protein was identified by Western blot. The specific serum IgG and fecal IgA was detected with ELISA.ResultsThe vp1 gene was 891bp and its sequence was consistent with that respected. Both PCR and restriction enzyme analysis showed that recombinant plasmid pBEX-VP1-LTB contained the target fusion gene. The expression product with a relative molecular mass of 73kD existed mostly in the form of inclusion body. Western-blot showed that the protein has a reactionogenicity with antibody of vp1-positive sera of rabbits.The IgG and IgA levels of mice immunized with VP1-LTB were significantly higher than those of control mice. The recombinant plasmids were successfully transformed into Bifidobacterium by electroporation. Western-blot showed that the protein has a reactionogenicity with antibody of vp1-positive sera of rabbits. Specific IgG in blood serum and IgA in intestinal tract of SD rat were found with ELISA.ConclusionThe recombinant expression vector for the vp1-ltb fusion gene was successfully constructed with overlap PCR technique. This recombinant expression vector was expressed both in E.coli BL21 (DE3) and Bifidobacterium. The recombinant protein showed good antigenicity and immunogenicity. This work paved the way for developing intramolecular adjuvant enginered vaccine against HFMD infected by EV71. The recombinant plasmid pBEX-VP1-LTB can express the target VP1-LTB with a reactionogenicity. The recombinant Bifidobacterium expressing VP1-LTB can induce specific membrana mucosa immune response which laid the foundation for the next research of an oral vaccine of EV71 with the Bifidobacterium expression system.