Recombinate An Oral Live Vaccine Of Rotavirus VP4 Based On Bifidobacterium Expresstion System

Rotavirus is the most important viral pathogen causing infantile diarrhea, more than a hundred million children infect it leading to a nearly a million death annually world wide. VP4,a kind of outer capsid protein,is one of the mainly antigens of rotavirus and takes important role in the infection of the host cell.Bifidobacterium exist normaly in human intestinal tract as 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 infant. It has some advantages of using Bifidobacterium as expression system for rotavirus oral vaccine development in safety, economic and convenience..Objective: We aimed to construct a shuttle expression plasmid of human rotavirus gene vp4 and express it in E.coli and B.infantis,then to analyze the immunicity of the recombined B.infantis by test the specific antibodies of VP4 in SD rats.Methods: (1) PCR product of vp4 was cloned into the expression vector pBES. Then the recombined plasmid was transformed into E.coli BL21 (DE3) and analysised by SDS-PAGE and Western-blot. (2) Transform the recombined plasmid into B.infantis by electroporation and analysised by SDS-PAGE and Western-blot. (3)Immunizing SD rat with recombined B.infantis by oral immunization and detecting the specific serum IgG and fecal sIgA with ELISA.Results: (1) The result was confirmed by enzyme digestion and sequencing analysis that human rotavirus vp4 was correctly cloned into pBES; PCR and restriction digestion shows that the recombined plasmid pBES-vp4 was transformed into B.infantis. (2) SDS-PAGE shows that a 87 KDa protein of VP4 was espressed in both E.coli BL21 (DE3) and the recombined B.infantis;Western-blot showed the protein was reactive with the antibody of human rotavirus . (3) ELISA indirectly proved that SD rats genetated Specific serum IgG and fecal sIgA.Conclusion: The recombined plasmid pBES-vp4 can express rotavirus VP4 protein in E.coli BL21(DE3) and B.infantis with reactiongenicity.The recombinant B.infantis expressing VP4 of human rotavirus can induce specific immune response which laid the foundation for the next research of an oral vaccine of rotavirus with the Bifidobacterium expression system.