Construction of an artificial recombinant bicistronic plasmid DNA vaccine against porcine rotavirus
2013
Tingting Cui | Jun Xiong | Yongzhi Wang | Xintian Wen | Xiaobo Huang | Yong Huang | Xiaoping Ma | Zhongkai Hu | Qin Zhao | Sanjie Cao
The attenuated <em>Salmonella typhimurium χ</em>4550 strain was used to harbour a reconstructed bicistronic DNA vaccine against porcine rotavirus, which carried the rotavirus nonstructural protein 4 (<em>NSP4</em>) 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 (<em>asd</em>) 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 <em>S. typhimurium χ</em>4550 strain by electrotransformation. An indirect immunofluorescence assay of the expressed COS-7 cell suggested that the recombinant <em>S. typhimurium χ</em>4550 strain was constructed successfully. The recombinant <em>S. typhimurium χ</em>4550 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.
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