Abstract
The attenuated Salmonella typhimurium χ4550 strain was used to harbour a reconstructed bicistronic DNA vaccine against porcine rotavirus, which carried the rotavirus nonstructural protein 4 (NSP4) and VP7 genes simultaneously. Using a balanced lethal system, the kanamycin resistance gene of expressing eukaryotic plasmids pVAX1 and pVAXD were replaced by the aspartate β-semialdehyde dehydrogenase (asd) gene. The NSP4 cleavage product (259-525) of rotavirus OSU strain and VP7 full-length genes were amplified by reverse transcription polymerase chain reaction and then inserted into the eukaryotic single-expression plasmid, pVAX1-asd, and the eukaryotic dual-expression plasmid, pVAXD-asd, respectively. The recombinant plasmids pVAX1-asd-NSP4, pVAX1-asd-VP7 and pVAXD-asd-NSP4-VP7 were transformed into the attenuated S. typhimurium χ4550 strain by electrotransformation. An indirect immunofluorescence assay of the expressed COS-7 cell suggested that the recombinant S. typhimurium χ4550 strain was constructed successfully. The recombinant S. typhimurium χ4550 strain was orally administered to BALB/c mice. The group immunised with dual- expression plasmids produced a significantly higher level of serum Immunoglobulin G (IgG) and intestinal Immunoglobulin A (IgA) than the group immunised with single-expression plasmids. These results indicated that eukaryotic bicistronic plasmid DNA vaccines could be successfully constructed to enhance humoural, mucosal and cellular immune response against rotavirus infection.
Highlights
Rotavirus is a highly infectious, triple-layered icosahedral, non-enveloped virus particle with a genome of 11 segments of double-stranded Ribonucleic acid (RNA) (Petrie, Estes & Graham 1983)
The asd segments with flat ends were ligated into plasmid pVAX1 and pVAXD and heat-shock transformated into χ6212 competent cells
We declare that the experiments were conducted in a reliable, authentic and valid manner. This experiment demonstrated that a bicistronic DNA vaccine that encodes the porcine rotavirus (PRV) VP7 protein and the C-terminal of the nonstructural protein 4 (NSP4) protein simultaneously and is delivered by an attenuated S. typhimurium χ4550 strain, was a potent vaccine, which induced a significantly higher level of serum Immunoglobulin G (IgG), mucosal Immunoglobulin A (IgA) and splenocyte IFN-γ than in the mice immunised with a single-expression vaccine
Summary
Rotavirus is a highly infectious, triple-layered icosahedral, non-enveloped virus particle with a genome of 11 segments of double-stranded Ribonucleic acid (RNA) (Petrie, Estes & Graham 1983). It is recognised as the most important cause of acute gastroenteritis and dehydration in young children and animals (Estes 2001). Currently available reassortant tetravalent rhesus rotavirus vaccines were found to cause a higher occurrence of intussusception in young children (Glass et al 2006). Effective protective rotavirus vaccines that are unlikely to cause intussusception would be highly desirable. It is necessary to develop an efficient method to increase the immunogenicity of DNA vaccines
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